Methods of stratifying patients for treatment with retinoic acid receptor-α agonists

ABSTRACT

Described herein are methods that define cellular populations that are sensitive to RARA agonists and identify patient populations that will benefit from treatment with RARA agonists. The methods may comprise administering RARA agonists to patient populations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/828,719 filed Dec. 1, 2017, now U.S. Pat. No. 10,240,210, which is acontinuation of U.S. application Ser. No. 15/582,311 filed Apr. 28,2017, now U.S. Pat. No. 9,868,994, which is a continuation ofInternational Application No. PCT/US17/26657 filed Apr. 7, 2017, whichclaims the benefit of and priority to U.S. provisional patentapplication Ser. No. 62/320,352, filed Apr. 8, 2016, the disclosures ofeach of which are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Retinoids are a class of compounds structurally related to vitamin A,comprising natural and synthetic compounds. Several series of retinoidshave been found clinically useful in the treatment of dermatological andoncological diseases. Retinoic acid and its other naturally occurringretinoid analogs (9-cis retinoic acid, all-trans 3,4-didehydro retinoicacid, 4-oxo retinoic acid and retinol) are pleiotropic regulatorycompounds that modulate the structure and function of a wide variety ofinflammatory, immune and structural cells. They are important regulatorsof epithelial cell proliferation, differentiation, and morphogenesis inlungs. Retinoids exert their biological effects through a series ofhormone nuclear receptors that are ligand inducible transcriptionfactors belonging to the steroid/thyroid receptor super family.

The retinoid receptors are classified into two families, the retinoicacid receptors (RARs) and the retinoid X receptors (RXRs), eachconsisting of three distinct subtypes (α, β, and γ). Each subtype of theRAR gene family encodes a variable number of isoforms arising fromdifferential splicing of two primary RNA transcripts. All-trans retinoicacid is the physiological hormone for the retinoic acid receptors andbinds with approximately equal affinity to all the three RAR subtypes,but does not bind to the RXR receptors for which 9-cis retinoic acid isthe natural ligand. Retinoids have anti-inflammatory effects, alter theprogression of epithelial cell differentiation, and inhibit stromal cellmatrix production. These properties have led to the development oftopical and systemic retinoid therapeutics for dermatological disorderssuch as psoriasis, acne, and hypertrophic cutaneous scars. Otherapplications include the control of acute promyelocytic leukemia, adeno-and squamous cell carcinoma, and hepatic fibrosis.

A limitation in the therapeutic use of retinoids has stemmed from therelative toxicity observed with the naturally occurring retinoids,all-trans retinoic acid and 9-cis retinoic acid. These natural ligandsare non-selective in terms of RAR subtype and therefore have pleiotropiceffects throughout the body, which are often toxic.

Various retinoids have been described that interact selectively orspecifically with the RAR or RXR receptors or with specific subtypes (α,β, γ) within a class. RARA specific agonists have held high promise forthe treatment of cancers and many have entered human clinical trials.However, only one RARA specific agonist, tamibarotene, has ever beenapproved for the treatment of cancer. Moreover, tamibarotene is onlyapproved in Japan and only for the treatment of acute promyelocyticleukemia, despite trials in the US and Europe. The disconnect betweenthe theoretical efficacy of RARA agonists in cancer and the dearth ofregulatory approvals for such agents raises the question of why suchagonists are not effective and safe in humans. Therefore, there is aneed to better understand why RARA agonists have not met theirtherapeutic potential.

Recent advances in genomic technology and the understanding of generegulatory circuits has led to the discovery of super enhancers. Whereasmany genes in a given tissue or cancer type may be regulated by thepresence of enhancers in proximity to the gene coding region, a smallminority of these represent a highly asymmetric and disproportionatelylarge loading of transcriptional marks and machinery relative to allother active genes. Recent discoveries suggest that such enhancers aretied to genes of special relevance to the function and survival of thecell harboring them. As such, an association of a super enhancer with agene indicates the relative significance of said gene to the survival ofthat cell.

SUMMARY OF THE INVENTION

The present disclosure provides technologies for detecting one or moreIRF8 biomarkers (e.g., presence, level, form, and/or activity of one ormore IRF8 gene components or products, including for example IRF8 superenhancer strength, ordinal rank, or prevalence rank and IRF8 mRNA levelor prevalence rank). The present disclosure demonstrates that cells(e.g., cancer cells or cells from a subject suffering from non-APL AMLor MDS) containing one or more IRF8 biomarkers, wherein the IRF8biomarker is or comprises expression of one or more of elevated IRF8mRNA levels or a super enhancer associated with an IRF8 gene are moresusceptible to the effects of a RARA agonist, such as tamibarotene.

The various embodiments, aspects and alternatives of this inventionsolve the problem of defining which cellular populations are sensitiveto agonists of retinoic acid receptor alpha (“RARA”), identifyingpatient populations that will benefit from treatment with RARA agonists(e.g., stratifying patients for treatment with a RARA agonist;separating RARA agonist responders from non-responders) and providingtreatment therapies directed at such patient populations. The solutionis based, at least in part, upon our discovery that elevated expressionof one or more IRF8 biomarkers in certain cancer cells is indicativethat such cells will be substantially more responsive” than similarcells that do not have an elevated IRF8 biomarker to treatment with aRARA agonist (e.g., tamibarotene).

In some embodiments, the present disclosure relates to a method oftreating cancer (e.g., non-APL AML or MDS) in a subject (e.g., a human)based on the level of IRF8 mRNA in the subject's cancer cells, whereinthe method comprises a step of administering to the subject an amount ofa RARA agonist (e.g., tamibarotene) effective to treat the disease. Insome aspects of these embodiments, the level of IRF8 mRNA in thesubject's cancer cells is equal to or above a pre-determined thresholdlevel.

In some embodiments, the present disclosure relates to a method oftreating cancer, wherein the method comprises a step of administeringtamibarotene to a subject having a cancer, wherein the cancer isdetermined to have an IRF8 biomarker, wherein the IRF8 biomarker is orcomprises expression of one or more of elevated IRF8 mRNA levels or asuper enhancer associated with an IRF8 gene.

In some embodiments, the present disclosure relates to a methodcomprising a step of administering therapy to a subject determined notto express one or more of elevated RARA mRNA levels or a super enhancerassociated with a RARA gene; and not to express one or more of elevatedIRF8 mRNA levels or a super enhancer associated with an IRF8 gene,wherein the therapy does not include administration of tamibarotene.

In some embodiments, the present disclosure relates to a method oftreating cancer, the method comprising a step of administering therapyto a subject determined (a) not to express one or more of elevated RARAmRNA levels or not to have a super enhancer associated with a RARA genewhose strength and/or ordinal rank is above a pre-determined threshold;and (b) to express one or more of elevated IRF8 mRNA levels or a superenhancer associated with an IRF8 gene, wherein the therapy istamibarotene.

In some embodiments, the present disclosure relates to a method oftreating cancer, the method comprising a step of administering therapyto a subject determined not to express one or more of elevated RARA mRNAlevels or a super enhancer associated with a RARA gene; and to expressone or more of elevated IRF8 mRNA levels or a super enhancer associatedwith an IRF8 gene, wherein the therapy is tamibarotene.

In some embodiments, the present disclosure relates to a method oftreating cancer, the method comprising steps of receiving informationrelated to IRF8 mRNA level in a subject suffering from a cancer; andadministering to the subject tamibarotene if the information indicatesthe IRF8 mRNA level or super enhancer level is equal to or above that ofa reference. In some aspects, a reference is a pre-determined threshold.In some aspects, a pre-determined threshold is a cutoff value or aprevalence cutoff.

In some embodiments, the present disclosure relates to a method oftreating cancer, the method comprising steps of receiving informationrelated to the presence of a super enhancer associated with an IRF8 genein a subject suffering from a cancer; and administering to the subjecttamibarotene if the information indicates that a super enhancer isassociated with an IRF8 gene.

In some embodiments, the present disclosure relates to a method ofpredicting the efficacy of a RARA agonist in a treatment of a cancercomprising the steps of determining if the cancer comprises a cellhaving IRF8 mRNA level that is equal to or above that of a reference,wherein the IRF8 mRNA level is equal to or above that of a reference ispredictive of RARA agonist efficacy in the treatment. In some aspects, areference is a pre-determined threshold. In some aspects, apre-determined threshold is a cutoff value or a prevalence cutoff.

In some embodiments, the present disclosure relates to a method ofpredicting the efficacy of a RARA agonist in a treatment of a cancercomprising the steps of determining if, in a subject suffering from acancer, the cancer comprises a cell that has a super enhancer associatedwith an IRF8 gene, wherein the presence of a super enhancer associatedwith an IRF8 gene indicates efficacious treatment of the cancer with aRARA agonist.

In some embodiments, the present disclosure relates to a methodcomprising steps of obtaining a biological sample comprising cancercells from a subject suffering from cancer; and detecting in thebiological sample one or more of IRF8 mRNA level is equal to or abovethat of that of a reference; or a super enhancer associated with an IRF8gene. In some aspects, a reference is a pre-determined threshold. Insome aspects, a pre-determined threshold is a cutoff value or aprevalence cutoff.

In some embodiments, the present disclosure relates to a method ofdiagnosing, prognosing, or treating a subject suffering from a cancercomprising the steps of obtaining a sample of the cancer from thesubject; and determining in the sample one or more of an IRF8 mRNA levelor the presence of a super enhancer associated with an IRF8 gene in thesubject.

In some embodiments, the present disclosure relates to a method ofdiagnosing, prognosing, or treating a subject suffering from a cancercomprising the steps of obtaining a sample of the cancer from thesubject; determining in the sample IRF8 mRNA level or the presence of asuper enhancer associated with an IRF8 gene in the subject; andadministering a therapeutic composition comprising a RARA agonist if oneor more of (a) IRF8 mRNA level is equal to or above that of that of areference; or (b) a super enhancer associated with an IRF8 gene. In someaspects, a reference is a pre-determined threshold. In some aspects, apre-determined threshold is a cutoff value or a prevalence cutoff.

In some embodiments, the present disclosure relates to a methodcomprising detecting one or more of RARA mRNA level or the strength orordinal rank of a super enhancer associated with a RARA gene in abiological sample obtained from a subject with a cancer; and detectingone or more of IRF8 mRNA level or a super enhancer associated with anIRF8 gene in the biological sample if the biological sample does notexpress one or more of elevated RARA mRNA level equal to above that of areference or a super enhancer associated with a RARA gene which has astrength or ordinal rank that is equal to or above a pre-determinedthreshold. In some aspects, a reference is a pre-determined threshold.In some aspects, a pre-determined threshold is a cutoff value or aprevalence cutoff.

In some embodiments, the present disclosure relates to a methodcomprising detecting one or more of RARA mRNA level or the strength orordinal rank of a super enhancer associated with a RARA gene in abiological sample obtained from a subject with a cancer; and detectingone or more of IRF8 mRNA level or a super enhancer associated with anIRF8 gene in the biological sample if the biological sample does expressone or more of elevated RARA mRNA level equal to above that of areference or a strength or ordinal rank of a super enhancer associatedwith a RARA gene which is equal to or above a pre-determined threshold.

In some embodiments, the present disclosure relates to a methodcomprising detecting one or more of IRF8 mRNA level or a super enhancerassociated with an IRF8 gene in a biological sample obtained from asubject with a cancer; and detecting one or more of RARA mRNA level orthe strength or ordinal rank of a super enhancer associated with a RARAgene in the biological sample if the biological sample does not expressone or more of elevated IRF8 mRNA level equal to above that of areference or a super enhancer associated with an IRF8 gene.

In some embodiments, the present disclosure relates to a methodcomprising detecting one or more of IRF8 mRNA level or a super enhancerassociated with an IRF8 gene in a biological sample obtained from asubject with a cancer; and detecting one or more of RARA mRNA level orthe strength or ordinal rank of a super enhancer associated with a RARAgene in the biological sample if the biological sample does express oneor more of elevated IRF8 mRNA level equal to above that of a referenceor a super enhancer associated with an IRF8 gene.

In some embodiments, the present disclosure relates to a method ofdiagnosing and treating a human subject suffering from a diseaseselected from non-APL AML and MDS, the method comprising:

a. diagnosing whether the subject has a tamibarotene-sensitive form ofthe disease based on a level of IRF8 mRNA previously determined to bepresent in a sample of diseased cells from the subject; and

b. administering to the subject an amount of tamibarotene effective totreat the disease.

In some aspects of these embodiments, the level of IRF8 mRNA is equal toor above a pre-determined threshold.

In some aspects of any of the foregoing embodiments which comprise thetreatment of a subject with tamibarotene, the subject is administered acombination of tamibarotene and a second therapeutic agent.

In some embodiments, the present disclosure relates to a method oftreating a cancer selected from non-APL or MDS in a subject based upon alevel of RARA mRNA and or a level of IRF8 mRNA in the subject's cancercells, wherein the treatment comprises administering to the subject acombination of tamibarotene and a second therapeutic agent. In someaspects of these embodiments, the subject has a RARA mRNA level equal toor above a threshold value. In some aspects of these embodiments, thesubject has an IRF8 mRNA level equal to or above a threshold value. Insome aspects of these embodiments, the subject has both a RARA mRNAlevel equal to or above a threshold value and an IRF8 mRNA level equalto or above a threshold value. In some aspects of these embodiments, thesubject is suffering from non-APL AML.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts IRF8 mRNA levels in seven different AML cell lines. Celllines indicated by the red bars demonstrate substantial responsivenessto tamibarotene treatment. Cell lines indicated by the blue barsdemonstrate little or no responsiveness to tamibarotene treatment.

FIG. 2 shows correlation of tamibarotene anti-proliferative potency(EC₅₀ value, nM) with IRF8 mRNA levels as measured by RNA-seq. Note thatthe top-left point with IRF8 mRNA level=1 (log 10) and tamibarotene EC₅₀value imputed as 50 μM (non-responsive) represents data from 2 AML celllines with low IRF8 mRNA levels and no anti-proliferative response totamibarotene. The correlation of tamibarotene sensitivity with IRF8 mRNAlevels was highly significant (p=0.0001, Spearman's correlation,two-tailed).

FIG. 3 depicts a rank order graph of IRF8 mRNA level in individualpatient AML samples and AML cell lines as measured by RNA-Seq. The AMLcell lines PL21, which was the cell line that had the lowest IRF8 mRNAlevel of any responsive cell line, and Kasumi, which was the cell linethat had the highest IRF8 mRNA level of any cell line unresponsive totamibarotene, are indicated. In this population, a 25% prevalence cutoffis equal to a RNA-Seq TPM value of approximately log₂(7).

FIG. 4 depicts the correlation between IRF8 mRNA level and RARA mRNAlevel in non-APL AML cell lines tested for response to tamibarotene.

FIG. 5 depicts the correlation between IRF8 mRNA level and RARA mRNAlevel in a population of AML patient samples. The dotted lines representa 25% prevalence cutoff for each mRNA

FIG. 6 shows correlation of tamibarotene anti-proliferative potency withIRF8 enhancer strength in AML cell lines. Plot of AML cell linetamibarotene sensitivity (EC₅₀ value, nM) as a function of IRF8 RECOMBenhancer scores. Note, top-left point with IRF8 enhancer score=0 andtamibarotene EC₅₀ value imputed as 50 μM (non-responsive) representsresults of 3 AML cell lines with no detectable IRF8 enhancer peak and noanti-proliferative response to tamibarotene.

FIG. 7 depicts IRF8 enhancer strength in AML patient samples. Rank orderplot of IRF8 enhancer strength under the RECOMB scoring method for 66AML patient samples. Each bar represents the IRF8 enhancer strength fora single AML patient. The Y-axis demonstrates individual IRF8 enhancerstrength as a multiple of the cutoff (defined as 1.0, indicated bydotted line) between super-enhancers (>1.0) and typical enhancers(≤1.0). Patient samples above this threshold appear in white fill whilethose below are in black. 14 of the 66 patient samples (21% of thepopulation) exceed the 1.0 threshold and were deemed to have IRF8 SEs.

FIG. 8 shows correlation of IRF8 mRNA levels with IRF8 enhancer strengthin AML patient samples. Plot of IRF8 mRNA transcript abundance byquantile normalized RNA-seq (log₂ TPM; Y-axis) as a function of IRF8RECOMB enhancer strength (X-axis) in 49 primary patient samples (thosewith both enhancer and expression values). The Spearman Rho correlationestimate is ˜0.0.81, with a p-value of 2.2×10¹².

FIG. 9 shows distribution of IRF8 enhancer strength in AML cell lines.Plot of IRF8 enhancer strength under the RECOMB scoring method for 26AML cell lines. Each bar represents the IRF8 enhancer strength for asingle AML cell line. The Y-axis illustrates individual IRF8 enhancerstrength as a multiple of the cutoff (defined as 1.0, indicated bydotted line) between super-enhancers (>1.0) and typical enhancers(≤1.0). Cell lines above this threshold appear in white fill, whilethose below are in black. Nine of the 26 AML cell lines (34% of thepopulation) exceed the 1.0 threshold and were deemed to have IRF8 SEs.

FIG. 10 shows correlation of IRF8 mRNA levels with IRF8 enhancerstrength in AML cell lines. Plot of IRF8 mRNA transcript abundance byquantile normalized RNA-seq TPM (Y-axis) as a function of IRF8 RECOMBenhancer strength (X-axis) in all non-APL AML cell lines for which bothRNA-seq and ChIP-seq data was available. The Spearman Rho correlationestimate is ˜0.82, with a p-value ˜2×10⁻⁶.

FIG. 11 depicts the response, as measured by % CD45⁺ cells, to dailydosing of tamibarotene in two different patient-derived mouse xenograftAML models. FIG. 11 also depicts the % CD45⁺ cells in different organsand biological fluids, as well as the time of survival of the mousemodels.

FIG. 12 depicts the response, as measured by % CD45⁺ cells, to dailydosing of tamibarotene in two additional patient-derived mouse xenograftAML models. FIG. 12 also depicts the % CD45⁺ cells in different organsand biological fluids in those models, as well as the time of survivalin those models.

FIG. 13 depicts the IRF8 mRNA level and the RARA mRNA level in each ofthe four AML patient samples used in the xenograft experiments depictedin FIGS. 11 and 12. Only the tamibarotene-responsive AM8096, whichproduced a tamibarotene-responsive xenograft, demonstrated an IRF8 mRNAabove a 100 TPM threshold. AM8096 and AM5512 (which demonstrated someresponsiveness to tamibarotene) demonstrated a RARA mRNA level above a10 TPM threshold.

FIG. 14 depicts a rank ordering of IRF8 mRNA levels detected in avariety of AML cell lines, AML primary patient samples, normal bloodcells and AML PDXs.

FIG. 15 depicts isobolograms for combinations of tamibarotene andazacitidine in various AML cell lines. Asterisks indicate data pointsoutside the maxima of the isobologram.

FIG. 16 depicts isobolograms for combinations of tamibarotene andarsenic trioxide in various AML cell lines. Asterisks indicate datapoints outside the maxima of the isobologram.

FIG. 17 depicts isobolograms for combinations of tamibarotene and Ara-Cin various AML cell lines. Asterisks indicate data points outside themaxima of the isobologram.

FIG. 18 depicts isobolograms for combinations of tamibarotene anddaunorubicin in various AML cell lines. Asterisks indicate data pointsoutside the maxima of the isobologram.

FIG. 19 depicts isobolograms for combinations of tamibarotene andmethotrexate in various AML cell lines. Asterisks indicate data pointsoutside the maxima of the isobologram.

FIG. 20 depicts isobolograms for combinations of tamibarotene andidarubicin in various AML cell lines. Asterisks indicate data pointsoutside the maxima of the isobologram.

FIG. 21 depicts isobolograms for combinations of tamibarotene andsorafenib in various AML cell lines. Asterisks indicate data pointsoutside the maxima of the isobologram.

DEFINITIONS

In this application, unless otherwise clear from context, (i) the term“a” may be understood to mean “at least one”; (ii) the term “or” may beunderstood to mean “and/or”; (iii) the terms “comprising” and“including” may be understood to encompass itemized components or stepswhether presented by themselves or together with one or more additionalcomponents or steps; and (iv) the terms “about” and “approximately” maybe understood to permit standard variation as would be understood bythose of ordinary skill in the art; and (v) where ranges are provided,endpoints are included.

Those skilled in the art will appreciate that one or more chemicalcompounds whose structure is depicted herein may have one or moreisomeric (e.g., enantiomeric, diastereomeric, and geometric (orconformational)) and/or tautomeric forms; for example, R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. In some embodiments, teachingsincluded herein may be applicable to and/or encompass any and all suchforms. Therefore, unless otherwise stated, single stereochemical isomersas well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds may all be within thescope of the invention. Similarly, unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Furthermore, those skilled in the art will appreciatethat, in some embodiments, chemical structures depicted herein mayencompass compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds whose structure isidentical to that depicted except for replacement of hydrogen bydeuterium or tritium, or replacement of a carbon by a ¹³C- or¹⁴C-enriched carbon, are within the scope of this invention. In certainembodiments, such compounds may be useful, for example, as analyticaltools, as probes in biological assays, and/or as therapeutic agents inaccordance with the present invention.

Agonist: As used herein, the term “agonist” may be used to refer to anagent, condition, or event whose presence, level, degree, type, or formcorrelates with increased level or activity of another agent (i.e., theagonized agent). In general, an agonist may be or include an agent ofany chemical class including, for example, small molecules,polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or anyother entity that shows the relevant activating activity. In someembodiments, an agonist may be direct (in which case it exerts itsinfluence directly upon its target); in some embodiments, an agonist maybe indirect (in which case it exerts its influence by other than bindingto its target; e.g., by interacting with a regulator of the target, sothat level or activity of the target is altered).

Agonist Therapy: As used herein, the term “agonist therapy” refers toadministration of an agonist that agonizes a particular target ofinterest to achieve a desired therapeutic effect. In some embodiments,agonist therapy involves administering a single dose of an agonist. Insome embodiments, agonist therapy involves administering multiple dosesof an agonist. In some embodiments, agonist therapy involvesadministering an agonist according to a dosing regimen known or expectedto achieve the therapeutic effect, for example, because such result hasbeen established to a designated degree of statistical confidence, e.g.,through administration to a relevant population.

Antagonist: As used herein, the term “antagonist” may be used to referto an agent, condition, or event whose presence, level, degree, type, orform correlates with decreased level or activity of another agent (e.g.,the inhibited agent, or target). In general, an antagonist may be orinclude an agent of any chemical class including, for example, smallmolecules, polypeptides, nucleic acids, carbohydrates, lipids, metals,and/or any other entity that shows the relevant inhibitory activity. Insome embodiments, an antagonist may be direct (in which case it exertsits influence directly upon its target); in some embodiments, anantagonist may be indirect (in which case it exerts its influence byother than binding to its target; e.g., by interacting with a regulatorof the target, so that level or activity of the target is altered).

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Acute Promyelocytic Leukemia: As used herein, the term “AcutePromyelocytic Leukemia” or “APL” refers to a subtype of acutemyelogenous leukemia (“AML”) characterized by a genetic translocationbetween human chromosomes 15 and 17. Accordingly, the term “Non-APL AML”refers to any subtype of AML that is not characterized by such a genetictranslocation.

Biological sample: As used herein, the term “biological sample” refersto any sample obtained from an individual suffering from a disease to betreated by the methods of this invention, including tissue samples (suchas tissue sections and needle biopsies of a tissue); cell samples (e.g.,cytological smears (such as Pap or blood smears) or samples of cellsobtained by microdissection); bone marrow samples (either whole,complete cell fractions thereof, or subpopulations of cells therein); orcell fractions, fragments or organelles (such as obtained by lysingcells and separating the components thereof by centrifugation orotherwise). Other examples of biological samples include blood, serum,urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid,mucus, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgicalbiopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva,swabs (such as buccal swabs), or any material containing biomoleculesthat is derived from a first biological sample. In some aspects, abiological sample from a subject suffering from non-APL AML or MDS is abone marrow aspirate. In other aspects, a biological sample from asubject suffering from non-APL AML or MDS is a fractionated whole bloodsample. In more specific aspects, a biological sample from a subjectsuffering from non-APL AML or MDS is a PBMC fraction from the subject'swhole blood (a “PBMC sample”). In still more specific aspects, a PBMCsample from a subject suffering from non-APL AML or MDS is furtherenriched for specific blasts using various enrichment techniques such asantibody-linked bead enrichment protocols, fluorescent label cellsorting, or other techniques known in the art (an “enriched PBMCsample”). In some embodiments, as will be clear from context, the term“sample” refers to a preparation that is obtained by processing (e.g.,by removing one or more components of and/or by adding one or moreagents to) a primary sample. Such a “processed sample” may comprise, forexample nucleic acids or proteins extracted from a sample or obtained bysubjecting a primary sample to techniques such as amplification orreverse transcription of mRNA, isolation and/or purification of certaincomponents, etc.

Biomarker: As used herein, the term “biomarker” refers to an entitywhose presence, level, or form, correlates with a particular biologicalevent or state of interest, so that it is considered to be a “marker” ofthat event or state. To give but a few examples, in some embodiments, abiomarker may be or comprises a marker for a particular disease state orstage, or for likelihood that a particular disease, disorder orcondition may develop. In some embodiments, a biomarker may be orcomprise a marker for a particular disease or therapeutic outcome, orlikelihood thereof. Thus, in some embodiments, a biomarker ispredictive, in some embodiments, a biomarker is prognostic, in someembodiments, a biomarker is diagnostic, of the relevant biological eventor state of interest. A biomarker may be an entity of any chemicalclass. For example, in some embodiments, a biomarker may be or comprisea nucleic acid, a polypeptide, a lipid, a carbohydrate, a smallmolecule, an inorganic agent (e.g., a metal or ion), or a combinationthereof. In some embodiments, a biomarker is a cell surface marker. Insome embodiments, a biomarker is intracellular. In some embodiments, abiomarker is found outside of cells (e.g., is secreted or is otherwisegenerated or present outside of cells, e.g., in a body fluid such asblood, urine, tears, saliva, cerebrospinal fluid, etc. In someembodiments, term refers to a gene expression product that ischaracteristic of a particular tumor, tumor subclass, stage of tumor,etc. Alternatively or additionally, in some embodiments, a presence orlevel of a particular marker correlates with activity (or activitylevel) of a particular signaling pathway, for example that may becharacteristic of a particular class of tumors. The statisticalsignificance of the presence or absence of a biomarker may varydepending upon the particular biomarker. In some embodiments, detectionof a biomarker is highly specific in that it reflects a high probabilitythat the tumor is of a particular subclass. Such specificity may come atthe cost of sensitivity (e.g., a negative result may occur even if thetumor is a tumor that would be expected to express the biomarker). Insome embodiments, a biomarker may be or comprise an IRF8 biomarker(e.g., presence, level, form, and/or activity of one or more IRF8 genecomponents or products, including for example IRF8 super enhancerstrength, ordinal rank, or prevalence rank and IRF8 mRNA level orprevalence rank). In some embodiments, a biomarker may include a RARAbiomarker (e.g., one or more RARA biomarkers (e.g., presence, level,form, and/or activity of one or more RARA gene components or products,including for example RARA super enhancer strength, ordinal rank, orprevalence rank and RARA mRNA level or prevalence rank). In someembodiments a biomarker refers to a combination of one or morebiomarkers, such as IRF8 and RARA.

Cancer: As used herein, the term “cancer” refers to a malignant neoplasmor tumor (Stedman's Medical Dictionary, 25th ed.; Hensly ed.; Williams &Wilkins: Philadelphia, 1990). The terms “neoplasm” and “tumor” are usedherein interchangeably and refer to an abnormal mass of tissue whereinthe growth of the mass surpasses and is not coordinated with the growthof a normal tissue. A “malignant neoplasm” is generally poorlydifferentiated (anaplasia) and has characteristically rapid growthaccompanied by progressive infiltration, invasion, and destruction ofthe surrounding tissue. Furthermore, a malignant neoplasm generally hasthe capacity to metastasize to distant sites. In some embodiments acancer is any malignant neoplasm or tumor wherein an IRF8 biomarker iscorrelated with responsiveness to a RARA agonist such as tamibarotene.In some embodiments a cancer is acute myelocytic leukemia (AML). In someembodiments a cancer is non-APL AML.

Combination therapy: As used herein, the term “combination therapy”refers to those situations in which a subject is simultaneously exposedto two or more therapeutic regimens (e.g., two or more therapeuticagents). In some embodiments, two or more agents or may be administeredsimultaneously; in some embodiments, such agents may be administeredsequentially; in some embodiments, such agents are administered inoverlapping dosing regimens. In some embodiments, “administration” ofcombination therapy may involve administration of one or more agents toa subject receiving the other agents in the combination. For clarity,combination therapy does not require that individual agents beadministered together in a single composition (or even necessarily atthe same time), although in some embodiments, two or more active agents,entities, or moieties may be administered together in a combinationcomposition, or even in a combination compound (e.g., as part of asingle chemical complex or covalent entity).

Cutoff value: As used herein, the terms “cutoff” and “cutoff value”means a value measured in an assay that defines the dividing linebetween two subsets of a population (e.g., responders andnon-responders). Thus, a value that is equal to or higher than thecutoff value defines one subset of the population; and a value that islower than the cutoff value defines the other subset of the population.

Diagnostic information: As used herein, “diagnostic information” or“information for use in diagnosis” is information that is useful indetermining whether a patient has a disease, disorder or conditionand/or in classifying a disease, disorder or condition into a phenotypiccategory or any category having significance with regard to prognosis ofa disease, disorder or condition, or likely response to treatment(either treatment in general or any particular treatment) of a disease,disorder or condition. Similarly, “diagnosis” refers to providing anytype of diagnostic information, including, but not limited to, whether asubject is likely to have or develop a disease, disorder or condition,state, staging or characteristic of a disease, disorder or condition asmanifested in the subject, information related to the nature orclassification of a tumor, information related to prognosis and/orinformation useful in selecting an appropriate treatment. Selection oftreatment may include the choice of a particular therapeutic agent orother treatment modality such as surgery, radiation, etc., a choiceabout whether to withhold or deliver therapy, a choice relating todosing regimen (e.g., frequency or level of one or more doses of aparticular therapeutic agent or combination of therapeutic agents), etc.

Dosage form or unit dosage form: Those skilled in the art willappreciate that the term “dosage form” may be used to refer to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Typically, each suchunit contains a predetermined quantity of active agent. In someembodiments, such quantity is a unit dosage amount (or a whole fractionthereof) appropriate for administration in accordance with a dosingregimen that has been determined to correlate with a desired orbeneficial outcome when administered to a relevant population (i.e.,with a therapeutic dosing regimen). Those of ordinary skill in the artappreciate that the total amount of a therapeutic composition or agentadministered to a particular subject is determined by one or moreattending physicians and may involve administration of multiple dosageforms.

Dosing regimen: Those skilled in the art will appreciate that the term“dosing regimen” may be used to refer to a set of unit doses (typicallymore than one) that are administered individually to a subject,typically separated by periods of time. In some embodiments, a giventherapeutic agent has a recommended dosing regimen, which may involveone or more doses. In some embodiments, a dosing regimen comprises aplurality of doses each of which is separated in time from other doses.In some embodiments, individual doses are separated from one another bya time period of the same length; in some embodiments, a dosing regimencomprises a plurality of doses and at least two different time periodsseparating individual doses. In some embodiments, all doses within adosing regimen are of the same unit dose amount. In some embodiments,different doses within a dosing regimen are of different amounts. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount different from the first dose amount. In some embodiments, adosing regimen comprises a first dose in a first dose amount, followedby one or more additional doses in a second dose amount same as thefirst dose amount In some embodiments, a dosing regimen is correlatedwith a desired or beneficial outcome when administered across a relevantpopulation (i.e., is a therapeutic dosing regimen).

Effective amount: As used herein, an “effective amount” of a compounddescribed herein, such as of Formula (I) refers to an amount sufficientto elicit the desired biological response, i.e., treating the condition.As will be appreciated by those of ordinary skill in this art, theeffective amount of a compound described herein, such as of Formula (I)may vary depending on such factors as the desired biological endpoint,the pharmacokinetics of the compound, the condition being treated, themode of administration, and the age and health of the subject. Aneffective amount encompasses therapeutic and prophylactic treatment. Forexample, in treating cancer, an effective amount of an inventivecompound may reduce the tumor burden or stop the growth or spread of atumor.

Enhancer: As used herein, the term “enhancer” refers to a region ofgenomic DNA acting to regulate genes up to 1 Mbp away. An enhancer mayoverlap, but is often not composed of, gene coding regions. An enhanceris often bound by transcription factors and designated by specifichistone marks.

Hydrate: As used herein, the term “hydrate” refers to a compound whichis associated with water. Typically, the number of the water moleculescontained in a hydrate of a compound is in a definite ratio to thenumber of the compound molecules in the hydrate. Therefore, a hydrate ofa compound may be represented, for example, by the general formula RxH₂O, wherein R is the compound and wherein x is a number greater than 0.A given compound may form more than one type of hydrates, including,e.g., monohydrates (x is 1), lower hydrates (x is a number greater than0 and smaller than 1, e.g., hemihydrates (R.0.5H₂O)), and polyhydrates(x is a number greater than 1, e.g., dihydrates (R.2H₂O) andhexahydrates (R.6H₂O)).

IRF8 gene: As used herein, the term “IRF8 gene” refers to a genomic DNAsequence that encodes an interferon consensus sequence-binding proteinor splice variant thereof and specifically excludes gene fusions thatcomprise all or a portion of the IRF8 gene. In some embodiments, theIRF8 gene is located at chr16:85862582-85990086 in genome build hg19.

“Improve,” “increase” or “reduce”: As used herein or grammaticalequivalents thereof, indicate values that are relative to a referencemeasurement, such as a measurement in the same individual prior toinitiation of a treatment described herein, or a measurement in acontrol individual (or multiple control individuals) in the absence ofthe treatment described herein. In some embodiments, a “controlindividual” is an individual afflicted with the same form of disease orinjury as an individual being treated.

Messenger RNA transcript: As used herein, the term “messenger RNAtranscript” or mRNA refers to the RNA transcription product from the DNAsequence that include one or more of the gene coding region.

Ordinal rank: As used herein, the term “ordinal rank” of a specifiedvalue means the rank order of that value as compared to a set of othervalues. For example, an ordinal rank of 100 in terms of the strength ofa super enhancer associated with a RARA gene in a test cell as comparedto other super enhancers in the test cell means that 99 other superenhancers in the test cell had greater strength than the super enhancerassociated with a RARA gene.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition is or may be administered,e.g., for experimental, diagnostic, prophylactic, cosmetic, and/ortherapeutic purposes. Typical patients include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and/or humans). In someembodiments, a patient is a human. A human includes pre and post-natalforms. In some embodiments, a patient is suffering from or susceptibleto one or more disorders or conditions. In some embodiments, a patientdisplays one or more symptoms of a disorder or condition. In someembodiments, a patient has been diagnosed with one or more disorders orconditions

Pharmaceutically acceptable salt: As used herein, the term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al., describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 1977, 66, 1-19, incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid, or malonic acidor by using other methods known in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, MALAT1e, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, andaryl sulfonate.

Population: As used herein, the term “population” or “population ofsamples” means a sufficient number (e.g., at least 30, 40, 50 or more)of different samples that reasonably reflects the distribution of thevalue being measured in a larger group. Each sample in a population ofsamples may be a cell line, a biological sample obtained from a livingbeing (e.g., a biopsy or bodily fluid sample), or a sample obtained froma xenograft (e.g., a tumor grown in a mouse by implanting a cell line ora patient sample), wherein each sample is from a living being sufferingfrom or from a cell line or xenograft representing, the same disease,condition or disorder.

Prevalence cutoff: As used herein, the term “prevalence cutoff” for aspecified value (e.g., the strength of a super enhancer associated withan IRF8 gene) means the prevalence rank that defines the dividing linebetween two subsets of a population (e.g., responders andnon-responders). Thus, a prevalence rank that is equal to or higher(e.g., a lower percentage value) than the prevalence cutoff defines onesubset of the population; and a prevalence rank that is lower (e.g., ahigher percentage value) than the prevalence cutoff defines the othersubset of the population.

Prevalence rank: As used herein, the term “prevalence rank” for aspecified value (e.g., the strength of a super enhancer associated withan IRF8 gene) means the percentage of a population that are equal to orgreater than that specific value. For example a 35% prevalence rank forthe strength of a super enhancer associated with an IRF8 gene in a testcell means that 35% of the population have an IRF8 gene enhancer with astrength equal to or greater than the test cell.

Prognostic and predictive information: As used herein, the terms“prognostic information” and “predictive information” are used to referto any information that may be used to indicate any aspect of the courseof a disease or condition either in the absence or presence oftreatment. Such information may include, but is not limited to, theaverage life expectancy of a patient, the likelihood that a patient willsurvive for a given amount of time (e.g., 6 months, 1 year, 5 years,etc.), the likelihood that a patient will be cured of a disease, thelikelihood that a patient's disease will respond to a particular therapy(wherein response may be defined in any of a variety of ways).Prognostic and predictive information are included within the broadcategory of diagnostic information.

Rank ordering: As used herein, the term “rank ordering” means theordering of values from highest to lowest or from lowest to highest.

RARA gene: As used herein, the term “RARA gene” refers to a genomic DNAsequence that encodes a functional retinoic acid receptor-α andspecifically excludes gene fusions that comprise all or a portion of theRARA gene. In some embodiments, the RARA gene is located atchr17:38458152-38516681 in genome build hg19.

Reference: as used herein describes a standard or control relative towhich a comparison is performed. For example, in some embodiments, anagent, animal, individual, population, sample, sequence, or value ofinterest is compared with a reference or control agent, animal,individual, population, sample, sequence or value. In some embodiments,a reference or control is tested and/or determined substantiallysimultaneously with the testing or determination of interest. In someembodiments, a reference or control is a historical reference orcontrol, optionally embodied in a tangible medium. Typically, as wouldbe understood by those skilled in the art, a reference or control isdetermined or characterized under comparable conditions or circumstancesto those under assessment. Those skilled in the art will appreciate whensufficient similarities are present to justify reliance on and/orcomparison to a particular possible reference or control.

Response: As used herein, a response to treatment may refer to anybeneficial alteration in a subject's condition that occurs as a resultof or correlates with treatment. Such alteration may includestabilization of the condition (e.g., prevention of deterioration thatwould have taken place in the absence of the treatment), ameliorationof, delay of onset of, and/or reduction in frequency of one or moresymptoms of the condition, and/or improvement in the prospects for cureof the condition, etc. In some instances, a response may be a subject'sresponse; in some instances a response may be a tumor's response.

Solvate: As used herein, the term “solvate” refers to forms of thecompound that are associated with a solvent, usually by a solvolysisreaction. This physical association may include hydrogen bonding.Conventional solvents include water, methanol, ethanol, acetic acid,DMSO, THF, diethyl ether, and the like. The compounds described herein,such as of Formula (I) may be prepared, e.g., in crystalline form, andmay be solvated. Suitable solvates include pharmaceutically acceptablesolvates and further include both stoichiometric solvates andnon-stoichiometric solvates. In certain instances, the solvate will becapable of isolation, for example, when one or more solvent moleculesare incorporated in the crystal lattice of a crystalline solid.“Solvate” encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates, and methanolates.

Strength: As used herein, the term “strength” when referring to aportion of an enhancer or a super enhancer, as used herein means thearea under the curve of the number of H3K27Ac or other genomic markerreads plotted against the length of the genomic DNA segment analyzed.Thus, “strength” is an integration of the signal resulting frommeasuring the mark at a given base pair over the span of the base pairsdefining the region being chosen to measure.

Subject: As used herein, a “subject” to which administration iscontemplated is a human (e.g., a male or female of any age group, e.g.,a pediatric subject (e.g., infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult, or senior adult)).

Super Enhancer: As used herein, the term “super enhancer” refers to asubset of enhancers that contain a disproportionate share of histonemarks and/or transcriptional proteins relative to other enhancers in aparticular cell. Because of this, a gene regulated by a super enhanceris predicted to be of high importance to the function of that cell.Super enhancers are typically determined by rank ordering all of theenhancers in a cell based on strength and determining using availablesoftware such as ROSE (https://bitbucket.org/young_computation/rose),the subset of enhancers that have significantly higher strength than themedian enhancer in the cell (see, e.g., U.S. Pat. No. 9,181,580, whichis herein incorporated by reference.

Threshold: As used herein, the terms “threshold” and “threshold level”mean a level that defines the dividing line between two subsets of apopulation (e.g., responders and non-responders). A threshold orthreshold level may be a prevalence cutoff or a cutoff value.

Treatment: As used herein, the terms “treatment,” “treat,” and“treating” refer to reversing, alleviating, delaying the onset of, orinhibiting the progress of a “pathological condition” (e.g., a disease,disorder, or condition, or one or more signs or symptoms thereof)described herein. In some embodiments, “treatment,” “treat,” and“treating” require that signs or symptoms of the disease disorder orcondition have developed or have been observed. In other embodiments,treatment may be administered in the absence of signs or symptoms of thedisease or condition (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example, to delay or preventrecurrence.

As used herein, the terms “condition,” “disease,” and “disorder” areused interchangeably.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

RARA and IRF8

The retinoic acid receptor subtype alpha (RARA) is a nuclear hormonereceptor that acts as a transcriptional repressor when unbound or boundby an antagonist, and as a gene activator in the agonist-bound state.The natural ligand of RARA is retinoic acid, also known as all-transretinoic acid (ATRA), which is produced from vitamin A.

Super-enhancers (SEs) are large, highly-active chromatin regions thatregulate key cell identity genes, including oncogenes in malignantcells. Using a gene control platform, we identified SEs genome-wide in60 primary AML patient samples to enable the discovery of novel tumorvulnerabilities. One of the SEs that exhibited a differential presenceamong patient samples was associated with the RARA gene encoding RARA.

Studies have demonstrated good correlations between tamibaroteneresponsiveness and either or both of RARA super enhancer strength andmRNA levels. However, for each of these potential RARA biomarkers, therewas a middle range within which tamibarotene responsiveness was mixed.The present disclosure provides insights and technologies that helpresolve such equivocal responsiveness results, and provides variouscompositions and methods useful in, among other things, characterizing,identifying, selecting, or stratifying patients based on likelyresponsiveness to tamibarotene therapy. For example, the presentdisclosure provides technologies that embody, define and/or utilize oneor more IRF8 biomarkers (e.g., presence, level, form, and/or activity ofone or more IRF8 gene components or products, including for example IRF8super enhancer strength, ordinal rank, prevalence rank, or IRF8 mRNAlevels), and demonstrates their usefulness in cancer therapy.

Using various AML cell lines and patient samples previously analyzed forstrength and ordinal of RARA enhancers, RARA mRNA levels andresponsiveness to tamibarotene, we looked for additional biomarkers thatwould correlate with responsiveness to tamibarotene. Interferon responsefactor 8 (IRF8) mRNA levels were found to be upregulated in similarpatient populations as RARA. IRF8 is an interferon responsivetranscription factor known to be critical to hematopoiesis and whosesignaling loss causes aberrant expansion of immature myeloid cells. InAML, IRF8 overexpression is observed and may correlate with poorclinical outcome. Despite this upregulation, IRF8 signaling is actuallyimpaired by repressive transcriptional cofactors and potentially RARAwhen it is in a SE-driven repressive state. Furthermore, interferon-αitself, the upstream signaling ligand for IRFs, exhibitspro-differentiation effects in AML and signaling cross-talk with theRARA pathway.

The present disclosure describes genome-wide expression and enhancerlevel analysis of a panel of AML patient tumor samples and cell lines toexamine the correlation of IRF8 gene enhancer strength, IRF8 mRNAlevels, and sensitivity to tamibarotene. The panel of AML cell lines waspreviously tested for and shown to have a correlation between itssensitivity to the anti-proliferative effects of the RARA agonisttamibarotene and both RARA enhancer strength and RARA mRNA levels. Inthis application we demonstrate that IRF8 mRNA levels are also elevatedin AML cell lines and AML patient samples that have elevated RARA mRNAlevels and that there is a correlation between IRF8 mRNA levels andresponsiveness to a RARA agonist, such as tamibarotene. We alsodemonstrate that there is a correlation between IRF8 enhancer strength(e.g., the presence of super-enhancer associated with IRF8), IRF8 mRNAlevels, RARA mRNA levels and responsiveness to tamibarotene. Thus, IRF8enhancer strength or IRF8 mRNA levels may be used alone, or inconjunction with RARA enhancer strength or RARA mRNA levels to identifypatients that will be responsive to treatment with a RARA agonist, suchas tamibarotene.

IRF8 and RARA Super-Enhancer Identification and Determination ofThreshold Levels

The identification of an enhancer or super enhancer may be achieved byvarious methods known in the art, for example as described in Cell 2013,155, 934-947 and PCT/US2013/066957, both of which are incorporatedherein by reference. In some embodiments, the identification of a superenhancer is achieved by obtaining cellular material and DNA from acancer sample in a patient (e.g., from a biopsy). The important metricsfor enhancer measurement occur in two dimensions—the length of the DNAover which genomic markers (e.g., H3K27Ac) are contiguously detected—andthe compiled incidence of genomic marker at each base pair along thatspan of DNA constituting the magnitude. The measurement of the areaunder the curve (“AUC”) resulting from integration of length andmagnitude analysis determines the strength of the enhancer. It is thestrength of the IRF8 or RARA super enhancer relative to a control thatis used in one aspect of the present invention to determine whether ornot a subject will be responsive to a RARA agonist (e.g., tamibarotene).It will be readily apparent to those of skill in the art, in view of theinstant specification, that if the length of DNA over which the genomicmarkers is detected is the same for both IRF8 or RARA and the control,then the ratio of the magnitude of the IRF8 or RARA super enhancerrelative to the control will be equivalent to the strength and may alsobe used to determine whether or not a subject will be responsive to aRARA agonist. In some embodiments, the strength of the IRF8 or RARAenhancer in a cell is normalized before comparing to other samples.Normalization is achieved by comparison to a region in the same cellknown to comprise a ubiquitous super-enhancer or enhancer that ispresent at similar levels in all cells. One example of such a ubiquitoussuper-enhancer region is the MALAT1 super-enhancer locus(chr11:65263724-65266724) (genome build hg19).

It has been determined through H3K27Ac ChIP-seq methods that there is asuper-enhancer locus associated with the RARA gene atchr17:38458152-38516681 (genome build hg19) and that there is asuper-enhancer locus associated with the IRF8 gene atchr16:85862582-85990086 (genome build hg19).

ChIP-sequencing, also known as ChIP-seq, is used to analyze proteininteractions with DNA. ChIP-seq combines chromatin immunoprecipitation(ChIP) with massively parallel DNA sequencing to identify the bindingsites of DNA-associated proteins. It can be used to map global bindingsites precisely for any protein of interest. Previously, ChIP-on-chipwas the most common technique utilized to study these protein—DNArelations. Successful ChIP-seq is dependent on many factors includingsonication strength and method, buffer compositions, antibody quality,and cell number.; see, e.g., T. Furey, Nature Reviews Genetics 13,840-852 (December 2012); M. L. Metzker, Nature Reviews Genetics 11,31-46 (January 2010); and P. J Park, Nature Reviews Genetics 10, 669-680(October 2009)). Genomic markers other that H3K27Ac that can be used toidentify super enhancers using ChIP-seq include, P300, CBP, BRD2, BRD3,BRD4, components of the mediator complex (J Loven, et al., Cell,153(2):320-334, 2013), histone 3 lysine 4 monomethylated (H3K4mel), orother tissue specific enhancer tied transcription factors (E Smith & AShilatifard, Nat Struct Mol Biol, 21(3):210-219, 2014) (S Pott & JasonLieb, Nature Genetics, 47(1):8-12, 2015).

In some embodiments, H3K27Ac or other marker ChIP-seq datasuper-enhancer maps of the entire genome of a cell line or a patientsample already exist. In some embodiments, one would simply determinewhether the strength, or ordinal rank of the enhancer or super-enhancerin such maps at the chr17:38458152-38516681 (genome build hg19) locuswas equal to or above the pre-determined threshold level. In someembodiments, one would simply determine whether the strength, or ordinalrank of the enhancer or super-enhancer in such maps at thechr16:85862582-85990086 (genome build hg19) locus was equal to or abovethe pre-determined threshold level.

It should be understood that the specific chromosomal location of IRF8,RARA, and MALAT1 may differ for different genome builds and/or fordifferent cell types. However, one of skill in the art, in view of theinstant specification, can determine such different locations bylocating in such other genome builds specific sequences corresponding tothe RARA and/or MALAT1 loci in genome build hg 19.

Other methods for identifying super enhancers include chromatinimmunoprecipitation (J E Delmore, et al., Cell, 146(6)904-917, 2011) andchip array (ChIP-chip), and chromatin immunoprecipitation followed byqPCR (ChIP-qPCR) using the same immunoprecipitated genomic markers andoligonucleotide sequences that hybridize to the chr17:38458152-38516681(genome build hg19) RARA locus or chr16:85862582-85990086 (genome buildhg19) IRF8 locus. In the case of ChIP-chip, the signal is typicallydetected by intensity fluorescence resulting from hybridization of aprobe and input assay sample as with other array based technologies. ForChIP-qPCR, a dye that becomes fluorescent only after intercalating thedouble stranded DNA generated in the PCR reaction is used to measureamplification of the template.

In some embodiments, determination of whether a cell has an IRF8 superenhancer strength equal to or above a requisite threshold level isachieved by comparing IRF8 enhancer strength in a test cell to thecorresponding IRF8 strength in a population of cell samples, whereineach of the cell samples is obtained from a different source (e.g., adifferent subject, a different cell line, a different xenograft)reflecting the same disease to be treated. In some aspects of theseembodiments, only primary tumor cell samples from subjects are used todetermine the threshold level. In some aspects of these embodiments, atleast some of the samples in the population will have been tested forresponsiveness to a specific RARA agonist in order to establish: a) thelowest IRF8 enhancer strength of a sample in the population thatresponds to that specific RARA agonist (“lowest responder”); and,optionally, b) the highest IRF8 enhancer strength of a sample in thepopulation that does not respond to that specific RARA agonist (“highestnon-responder”). In these embodiments, a cutoff of IRF8 enhancerstrength above which a test cell would be considered responsive to thatspecific RARA agonist is set: i) equal to or up to 5% above the IRF8enhancer strength in the lowest responder in the population; or ii)equal to or up to 5% above the IRF8 enhancer strength in the highestnon-responder in the population; or iii) a value in between the IRF8enhancer strength of the lowest responder and the highest non-responderin the population.

It should be understood that in the above embodiments that not all ofthe samples in a population necessarily are to be tested forresponsiveness to a RARA agonist, but all samples are measured for IRF8enhancer strength and/or IRF8 mRNA levels. In some embodiments, thesamples are rank ordered based on IRF8 enhancer strength. The choice ofwhich of the three methods set forth above to use to establish thecutoff will depend upon the difference in IRF8 enhancer strength betweenthe lowest responder and the highest non-responder in the population andwhether the goal is to minimize the number of false positives or tominimize the chance of missing a potentially responsive sample orsubject. When the difference between the lowest responder and highestnon-responder is large (e.g., when there are many samples not tested forresponsiveness that fall between the lowest responder and the highestnon-responder in a rank ordering of IRF8 enhancer strength), the cutoffis typically set equal to or up to 5% above the IRF8 enhancer strengthin the lowest responder in the population. This cutoff maximizes thenumber of potential responders. When this difference is small (e.g.,when there are few or no samples untested for responsiveness that fallbetween the lowest responder and the highest non-responder in a rankordering of IRF8 enhancer strength), the cutoff is typically set to avalue in between the IRF8 enhancer strength of the lowest responder andthe highest non-responder. This cutoff minimizes the number of falsepositives. When the highest non-responder has an IRF8 enhancer strengththat is greater than the lowest responder, the cutoff is typically setto a value equal to or up to 5% above the IRF8 enhancer strength in thehighest non-responder in the population. This method also minimizes thenumber of false positives.

In some embodiments, determination of whether a cell has an IRF8 superenhancer equal to or above a requisite threshold level is achieved bycomparing the ordinal of IRF8 enhancer strength in a test cell to theordinal of IRF8 enhancer strength in a population of cell samples,wherein each of the cell samples is obtained from a different source(e.g., a different subject, a different cell line, a differentxenograft). In these embodiments, at least some of the samples in thepopulation will have been tested for responsiveness to a specific RARAagonist in order to establish: a) the lowest IRF8 enhancer strengthordinal of a sample in the population that responds to that specificRARA agonist (“lowest ordinal responder”); and, optionally, b) thehighest IRF8 enhancer strength ordinal of a sample in the populationthat does not respond to that specific RARA agonist (“highest ordinalnon-responder”). In these embodiments, a cutoff of IRF8 enhancerstrength ordinal above which a test cell would be considered responsiveto that specific RARA agonist is set: i) equal to or up to 5% above theIRF8 enhancer strength ordinal in the lowest ordinal responder in thepopulation; or ii) equal to or up to 5% above the IRF8 enhancer strengthordinal in the highest ordinal non-responder in the population; or iii)a value in between the IRF8 enhancer strength ordinal of the lowestordinal responder and the highest ordinal non-responder in thepopulation.

It should be understood in the above embodiments, that typically not allof the samples in a population need to be tested for responsiveness to aRARA agonist, but all samples are measured for IRF8 enhancer strengthand the ordinal of IRF8 enhancer strength compared to other enhancers inthe same sample is established. The ordinal is typically obtained bymeasuring the strength of all other enhancers in the cell anddetermining what rank (e.g., the ordinal) in terms of strength the IRF8enhancer has as compared to the other enhancers.

In some embodiments, the samples are rank ordered based on the ordinalof IRF8 enhancer strength. The choice of which of the three methods setforth above to use in order to establish the cutoff will depend upon thedifference in ordinal of IRF8 enhancer strength between the lowestordinal responder and the highest ordinal non-responder in thepopulation and whether the cutoff is designed to minimize falsepositives or maximize the number of responders. When this difference islarge (e.g., when there are many samples not tested for responsivenessthat fall between the lowest ordinal responder and the highest ordinalnon-responder in a rank ordering of ordinals of IRF8 enhancer strength),the cutoff is typically set equal to or up to 5% above the ordinal ofIRF8 enhancer strength in the lowest ordinal responder in thepopulation. When this difference is small (e.g., when there are few orno samples untested for responsiveness that fall between the lowestordinal responder and the highest ordinal non-responder in a rankordering of ordinal of IRF8 enhancer strength), the cutoff is typicallyset to a value in between the ordinal of IRF8 enhancer strength of thelowest ordinal responder and the highest ordinal non-responder. When thehighest ordinal non-responder has an ordinal of IRF8 enhancer strengththat is greater than that of the lowest responder, the cutoff istypically set to a value equal to or up to 5% above the ordinal of IRF8enhancer strength in the highest ordinal non-responder in thepopulation.

In some embodiments where a test cell or sample is compared to apopulation, the cutoff value(s) obtained for the population (e.g., IRF8enhancer strength or IRF8 enhancer ordinal) is converted to a prevalencerank and the cutoff is expressed as a percent of the population havingthe cutoff value or higher, e.g., a prevalence cutoff. Without beingbound by theory, applicants believe that the prevalence rank of a testsample will be similar regardless of the methodology used to determineIRF8 enhancer strength. Thus, a prevalence cutoff determined for oneparameter (e.g., IRF8 enhancer strength ordinal) is portable and can beapplied to another parameter (e.g., IRF8 mRNA level) to determine thecutoff value for that other parameter. This allows the determination ofa cutoff value for any parameter without having to experimentallydetermine the correlation between levels of such parameter andresponsiveness to a RARA agonist. All that needs to be determined iswhat level of such other parameter corresponds to the prior determinedprevalence cutoff in a population.

In some embodiments, the methods discussed above can be employed tosimply determine if a diseased cell from a subject has a super enhancerassociated with an IRF8 gene. In these embodiments, the presence of anIRF8-associated super enhancer indicates that the subject will respondto a RARA agonist. In one aspect of these embodiments, the cell isdetermined to have a super enhancer associated with an IRF8 gene whenthe IRF8-associated enhancer has a strength that is equal to or abovethe enhancer associated with MALAT-1. In alternate aspects of theseembodiments, the cell is determined to have a super enhancer associatedwith an IRF8 gene when the IRF8-associated enhancer has a strength thatis at least 10-fold greater than the median strength of all of theenhancers in the cell. In other alternate aspects of these embodiments,the cell is determined to have a super enhancer associated with an IRF8gene when the IRF8-associated enhancer has a strength that is above thepoint where the slope of the tangent is 1 in a rank-ordered graph ofstrength of each of the enhancers in the cell.

In some embodiments, the methods discussed above can be employed toadditionally determine if a diseased cell from a subject expresses asuper enhancer associated with a RARA gene that has a strength, ordinalrank, or prevalence rank that is equal to or above a pre-determinedthreshold level. In some aspects of these embodiments, a determinationthat either: a) the diseased cell has a super enhancer associated with aIRF8 gene (or that such super enhancer has a strength or ordinal rankthat is equal to or above a pre-determined threshold level; or b) thediseased cell has a super enhancer associated with a RARA gene that hasa strength or ordinal rank that is equal to or above a pre-determinedthreshold level indicates that the subject will respond to a RARAagonist. In other aspects of these embodiments, a determination that: a)the diseased cell has a super enhancer associated with a IRF8 gene (orthat such super enhancer has a strength or ordinal rank that is equal toor above a pre-determined threshold level; and b) the diseased cell hasa super enhancer associated with a RARA gene that has a strength orordinal rank that is equal to or above a pre-determined threshold levelindicates that the subject will respond to a RARA agonist.

IRF8 mRNA Level Determination

In some embodiments, IFR8 mRNA levels may be used instead ofsuper-enhancer strength or ordinal rank to determine sensitivity to aRARA agonist. IRF8 mRNA may be quantified and correlates very well withsuper-enhancer strength at that locus (FIG. 10). We have determined thatmRNA transcripts encoding IRF8 correlate with sensitivity to RARAagonists (FIG. 8), and thus in some embodiments, IRF8 mRNA levels can beused to identify cells that will respond to RARA agonists.

In some embodiments, sequences of one or more biomarkers (e.g.,epigenetic markers such a transcript level) are assessed. In someembodiments, DNA sequencing may be used to determine the sequence ofindividual genes, larger genetic regions (e.g. clusters of genes oroperons), full chromosomes or entire genomes. In some embodiments, RNAsequencing may be used. One of skill in the art would understand variousmethods available to determine sequences of individual genes, largergenetic regions (e.g. clusters of genes or operons), full chromosomes orentire genomes. In some embodiments, next-generation sequencing may beused. In some embodiments, next-generation sequencing of full genomesmay be used. In some embodiments, sequencing may be utilized to quantifylevel of transcript.

In some embodiments, IRF8 mRNA levels in a subject (e.g., in a tumorsample, in a cancer cell sample, in a blood sample, etc.) are compared,using the same assay, to the IRF8 mRNA levels in a population ofsubjects having the same disease or condition to identify RARA agonistresponders. In these embodiments, at least some of the samples in thepopulation will have been tested for responsiveness to a specific RARAagonist in order to establish: a) the lowest IRF8 mRNA level of a samplein the population that responds to that specific RARA agonist (“lowestmRNA responder”); and, optionally, b) the highest IRF8 mRNA level of asample in the population that does not respond to that specific RARAagonist (“highest mRNA non-responder”). In these embodiments, a cutoffof IRF8 mRNA level above which a test cell would be consideredresponsive to that specific RARA agonist is set: i) equal to or up to 5%above the IRF8 mRNA level in the lowest mRNA responder in thepopulation; or ii) equal to or up to 5% above the IRF8 mRNA level in thehighest mRNA non-responder in the population; or iii) a value in betweenthe IRF8 mRNA level of the lowest mRNA responder and the highest mRNAnon-responder in the population.

In some embodiments not all of the samples in a population need to betested for responsiveness to a RARA agonist, but all samples aremeasured for IRF8 mRNA levels. In some embodiments, the samples are rankordered based on IRF8 mRNA levels. The choice of which of the threemethods set forth above to use to establish the cutoff will depend uponthe difference in IRF8 mRNA levels between the lowest mRNA responder andthe highest mRNA non-responder in the population and whether the cutoffis designed to minimize false positives or maximize the potential numberof responders. When this difference is large (e.g., when there are manysamples not tested for responsiveness that fall between the lowest mRNAresponder and the highest mRNA non-responder in a rank ordering of IRF8mRNA levels), the cutoff is typically set equal to or up to 5% above theIRF8 mRNA level in the lowest mRNA responder in the population. Whenthis difference is small (e.g., when there are few or no samplesuntested for responsiveness that fall between the lowest mRNA responderand the highest mRNA non-responder in a rank ordering of IRF8 mRNAlevels), the cutoff is typically set to a value in between the IRF8 mRNAlevels of the lowest mRNA responder and the highest mRNA non-responder.When the highest mRNA non-responder has an IRF8 mRNA level that isgreater than the lowest mRNA responder, the cutoff is typically set to avalue equal to or up to 5% above the IRF8 mRNA levels in the highestmRNA non-responder in the population.

In some embodiments, the population is rank ordered based on IRF8 mRNAlevel. In these embodiments, the IRF8 mRNA level in each sample ismeasured and compared to the mRNA levels of all other mRNAs in the cellto obtain an ordinal ranking of the IRF8 mRNA level. A cutoff based onIRF8 mRNA ordinal ranking is then determined based on samples in thepopulation tested for responsiveness to a RARA agonist in the samemanner as described previously for determining an IRF8 super enhancerstrength ordinal cutoff. The determined IRF8 mRNA ordinal cutoff is thenused either directly or to determine a prevalence cutoff, either ofwhich is then used to stratify additional samples for potentialresponsiveness to a RARA agonist.

In some embodiments, the cutoff for IRF8 mRNA levels is determined usingthe prevalence cutoff established based on IRF8 enhancer strength orIRF8 enhancer strength ordinal, as described above. In some aspects ofthese embodiments, a population is measured for mRNA levels and theprior determined prevalence cutoff is applied to that population todetermine an mRNA cutoff level. In some aspects of these embodiments arank-order standard curve of IRF8 mRNA levels in a population iscreated, and the pre-determined prevalence cutoff is applied to thatstandard curve to determine the IRF8 mRNA cutoff level.

In some aspects of embodiments where a test cell or sample is comparedto a population, the cutoff mRNA level value(s) obtained for thepopulation is converted to a prevalence rank and the mRNA level cutoffis expressed as a percent of the population having the cutoff value orhigher, e.g., a prevalence cutoff.

Without being bound by theory, applicants believe that the prevalencerank of a test sample and the prevalence cutoff in a population will besimilar regardless of the methodology used to determine IRF8 mRNAlevels.

In some aspects of these embodiments, a subject is identified as a RARAagonist responder if its IRF8 mRNA level corresponds to a prevalencerank in a population of about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%,72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%,58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%,44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%,30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20% as determinedby IRF8 mRNA levels in the population. In some embodiments, the cutoffvalue is established based on the prevalence cutoff established for IRF8enhancer strength. In some embodiments, the cutoff value is establishedbased on the prevalence cutoff established for IRF8 enhancer strengthordinal. In some embodiments, the cutoff value is established based onIRF8 mRNA levels. In some embodiments, a cutoff value for AML, non-APLAML, or MDS patients is established based on the prevalence valuedetermined for IRF8 enhancer strength ordinal, and that prevalence valueis used to determine the cutoff value for IRF8 mRNA levels. In someembodiments, the cutoff value for AML, non-APL AML or MDS patients isdetermined using a prevalence cutoff of between about 20-45% (e.g.,between about 20-25%, 25-30%, 25-35%, 25-40%, 20-30%, 20-35%, 20-40%,20-45%, 21-34%, 22-34%, 25-34%, 21-25%, 22-25%, 23-25%, 24-25%, or21-22%). In some embodiments, the cutoff value for AML, non-APL AML orMDS patients is determined using a prevalence value of 34%. In someembodiments, the cutoff value for AML, non-APL AML or MDS patients isdetermined using a prevalence value of 25%. In some embodiments, thecutoff value for AML, non-APL AML or MDS patients is determined using aprevalence value of 22%. In some embodiments, the cutoff value for AML,non-APL AML or MDS patients is determined using a prevalence value of21%.

In still other embodiments, a population may be divided into threegroups—responders, partial responders and non-responders and two cutoffvalues or prevalence cutoffs are set. The partial responder group mayinclude responders and non-responders, as well as those populationmembers whose response to a RARA agonist was not as high as theresponder group. In these embodiments, two cutoff values or prevalencecutoffs are determined. This type of stratification may be particularlyuseful when in a population the highest IRF8 mRNA non-responder has anIRF8 mRNA levels that is greater than the lowest RARA mRNA responder. Inthis scenario the cutoff level or prevalence cutoff between respondersand partial responders is set equal to or up to 5% above the IRF8 mRNAlevel of the highest IRF8 mRNA non-responder; and the cutoff level orprevalence cutoff between partial responders and non-responders is setequal to or up to 5% below the IRF8 mRNA level of the lowest IRF8 mRNAresponder. The determination of whether partial responders should beadministered a RARA agonist will depend upon the judgment of thetreating physician and/or approval by a regulatory agency.

Methods of quantifying specific RNA sequences in a cell or biologicalsample are known in the art and include, but are not limited to,fluorescent hybridization such as utilized in services and productsprovided by NanoString Technologies, array based technology(Affymetrix), reverse transcriptase qPCR as with SYBR® Green (LifeTechnologies) or TaqMan® technology (Life Technologies), RNA sequencing(e.g., RNA-seq), RNA hybridization and signal amplification as utilizedwith RNAscope® (Advanced Cell Diagnostics), or northern blot.

In some aspects of these embodiments, the level of RNA transcript(either mRNA or another RARA or IRF8 transcript) in both the test celland the control cell or all members of the population are normalizedbefore comparison. Normalization involves adjusting the determined levelof an IRF8 or RARA RNA transcript by comparison to either another RNAtranscript that is native to and present at equivalent levels in both ofthe cells (e.g., GADPH mRNA, 18S RNA), or to a fixed level of exogenousRNA that is “spiked” into samples of each of the cells prior tosuper-enhancer strength determination (J Lovén et al., Cell,151(3):476-82 (2012); J Kanno et al., BMC Genomics 7:64 (2006); J Van dePeppel et al., EMBO Rep 4:387-93 (2003)).

Cancers and Other Diseases

The methods of the present disclosure are useful to treat any cancerthat is characterized by the association of a super enhancer with IRF8or an IRF8 mRNA level that is equal to or above a threshold level insuch cancer. Super enhancer-associated IRF8 genes may be more prevalentin certain types of cancers than others. In some embodiments, superenhancer-associated IRF8 genes may be more prevalent in non-APL AML andin MDS than other cancers or pre-cancerous conditions.

In some embodiments, the disease to be treated in the methods of theinvention is cancer. In some embodiments, the disease to be treated isselected from non-APL AML and MDS. In some embodiments, the disease tobe treated is non-APL AML and MDS that is not characterized by achromosomal translocation involving an IRF8 gene.

In some embodiments, the subject to be treated with a RARA agonist(e.g., tamibarotene) is suffering from relapsed or refractory non-APLAML. A subject is classified as having relapsed or refractory non-APLAML if they: a) do not demonstrate a partial response after a firstcycle of induction chemotherapy; or b) do not demonstrate a completeresponse after a second cycle of induction chemotherapy; or c) relapseafter conventional chemotherapy; or d) relapse after undergoing a singlestem cell transplantation.

In some embodiments, the subject to be treated with a RARA agonist(e.g., tamibarotene) is suffering from refractory MDS. A subject isclassified as having refractory MDS if they: a) are categorized ashaving high risk or intermediate-2 MDS (as determined using theInternational Prognostic Staging System (“IPPS”)) and have failed toachieve any hematologic improvement (as measured by IWG 2006 criteria)after at least 4 cycles of induction therapy with hypomethylating agents(e.g., azacitidine, decitabine), or has relapsed after any duration ofcomplete or partial response; orb) are categorized as IPSSintermediate-1 or low-risk MDS and are either transfusion dependent orhave failed treatment with erythropoiesis stimulating agents (ESA).

In other embodiments, the subject to be treated with a RARA agonist(e.g., tamibarotene) is an elderly unfit subject. The term “elderlyunfit” as used herein means the subject is a human at least 60 years ofage and who is determined by a physician to not be a candidate forstandard induction therapy.

RARA Agonists

The choice of RARA agonist with which to treat a patient identified ashaving a super enhancer level may be made from any RARA agonist known inthe art. It is preferable that a RARA agonist utilized in the methods ofthe invention be specific for RARA and have significantly less (at least10× less, at least 100× less, at least 1,000× less, at least 10,000×less, at least 100,000× less) agonistic activity against other forms ofRaR, e.g., RaR-β and RaR-γ.

In some embodiments, a RARA agonist is selected from a compounddisclosed in or any compound falling within the genera set forth in anyone of the following United States patents: U.S. Pat. Nos. 4,703,110,5,081,271, 5,089,509, 5,455,265, 5,759,785, 5,856,490, 5,965,606,6,063,797, 6,071,924, 6,075,032, 6,187,950, 6,355,669, 6,358,995, and6,387,950, each of which is incorporated by reference.

In some embodiments, a RARA agonist is selected from any of thefollowing known RARA agonists set forth in Table 1, or apharmaceutically acceptable salt thereof, or a solvate or hydrate of theforegoing:

TABLE 1 Exemplary RARA Agonists useful in the invention. Structure CodeName(s)

Am-580; CD- 336; Ro-40- 6055

AM-80; INNO-507; NSC-608000; OMS-0728; TM-411; TOS- 80; TOS-80T; Z-208;tamibarotene

Am-555S; TAC-101; amsilarotene

ER-34617

ER-38930

ER-65250

ER-38925

ER-35368

E-6060

ER-41666

AGN-195183; NRX-195183; VTP-195183; VTP-5183 IRX-5183

BMS-228987

BMS-276393

BMS-231974

ABPN; CBG- 41

PTB

A-112

BD-4; BJ-1

Tazarotene; AGN-190168

Ch-55

In some embodiments, a RARA agonist is tamibarotene.

Therapeutic Regimens

Markers and Characterization

In some embodiments, technologies provided by the present disclosureinvolve assessment of type of cancer from which a patient is suffering.In some embodiments, a patient is suffering from non-APL acutemyelocytic leukemia (AML). In some embodiments, a patient is sufferingfrom or myelodysplastic syndrome (MDS).

In general, the present disclosure provides technologies according towhich one or more markers or characteristics of a subject is analyzedand/or assessed; in some embodiments, a therapeutic decision is madebased on such analysis and/or assessment.

In some embodiments, a marker is an agent or entity whose presence,form, level, and/or activity is correlated in a relevant population witha relevant feature (e.g., type or stage of cancer). In some embodiments,the present disclosure contemplates identification, classification,and/or characterization of one or more biomarkers relevant for thetreatment of non-APL AML with a RARA agonist. In some embodiments, thepresent disclosure contemplates identification, classification, and/orcharacterization of one or more biomarkers relevant for the treatment ofMDS with a RARA agonist.

In some embodiments, classification of a patient as suffering from aparticular type of cancer may involve assessment of stage of cancer. Insome embodiments, classification of a patient as suffering from aparticular type of cancer may involve assessment of disease burden inthe patient (e.g. the number of cancer cells, the size of a tumor,and/or amount of cancer in the body).

In general, type of cancer may be assessed in accordance with thepresent invention via any appropriate assay, as will be readilyappreciated by those of ordinary skill in the art. A variety of assaysfor cancer type are known in the art including, for example, those thatutilize histological assessment (e.g., of a biopsy sample), imaging(e.g., magnetic resonance imaging (MRI), positron emission tomography(PET), computed tomography (CT) ultrasound, endoscopy, x-rays (e.g.,mammogram, barium swallow, panorex), ductogram, or bone scan.

In some embodiments, RARA agonist therapy comprises assessing a level ofone or more biomarkers indicative of a stage or a form of non-APL AML orMDS. In some embodiments, RARA agonist therapy comprises assessing IRF8mRNA level and, optionally, RARA mRNA level. In some embodiments, RARAagonist therapy comprises the presence of a super enhancer associatedwith an IRF8 gene and, optionally, the strength or ordinal rank of asuper enhancer associated with a RARA gene. In some embodiments, RARAagonist therapy comprises assessing IRF8 mRNA level or the presence of asuper enhancer associated with an IRF8 gene and, optionally RARA mRNAlevel, the strength or ordinal rank of a super enhancer associated witha RARA gene.

Without being bound by theory, applicants believe that subsets of PBMCsthat have only one of CD34 or CD117 markers can also be used effectivelyto determine IRF8 mRNA or super-enhancer levels. Moreover, certain IRF8mRNA analysis techniques, such as RNAScope® do not require enriching aPBMC sample prior to analysis because those techniques provideanalytical enrichment of mRNA from the desired cells based on the use ofspecific oligonucleotide hybridization/amplification procedures.

Patient Populations

In some embodiments, RARA agonist therapy is administered in accordancewith the present disclosure to one or more patients (e.g., to a patientpopulation) as described herein.

In some embodiments, a patient population includes one or more subjects(e.g., comprises or consists of subjects) suffering from cancer. In someembodiments, a patient population includes one or more subjectssuffering from non-APL AML. In some embodiments, a patient populationincludes one or more subjects suffering from MDS.

In some embodiments, a patient population includes one or more subjects(e.g., comprises or consists of subjects) who received previous therapyfor treatment of cancer (e.g., non-APL AML or MDS). In some embodiments,a patient population includes one or more subjects (e.g., comprises orconsists of subjects) who have not received previous therapy fortreatment of cancer (e.g., non-APL AML or MDS). In some embodiments, apatient population comprises or consists of patients who have notreceived previous therapy for treatment of non-APL AML or MDS.

In some embodiments, a patient who received previous therapy may havereceived previous therapy selected from the group consisting ofchemotherapy, immunotherapy, radiation therapy, palliative care,surgery, and combinations thereof. In some embodiments, a patient hasreceived a transplant. In some embodiments, a patient has receivedstandard cytotoxic chemotherapy. In some embodiments, standard cytotoxicchemotherapy includes cytarabine and/or an anthracycline. In someembodiments, standard cytotoxic chemotherapy may include additionalchemotherapy and/or hematopoietic stem cell transplantation (HSTC). Insome embodiments, a patient has received hypomethylating agents. In someembodiments, a patient has received lenalidomide.

In some embodiments, a patient population includes one or more subjects(e.g., comprises or consists of subjects) who have received and/or arereceiving other therapy, e.g., so that a RARA agonist therapy (e.g.,tamibarotene) composition is administered in combination with the othertherapy (e.g. chemotherapy agents). In some embodiments, such othertherapy may comprise or consist of therapy for cancer (e.g., asdescribed herein), pain, nausea, constipation, for treatment of one ormore side effects (e.g., pruritus, hair loss, sleeplessness, etc.)associated with cancer therapy, etc., or any combination thereof. Thepresent invention provides a method of treating non-APL AML or MDS,which comprises treating a patient identified as having non-APL AML orMDS, with a therapeutically effective amount of RARA agonist therapy(e.g., tamibarotene) or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a method ofpreventing or delaying the onset of non-APL AML or MDS, comprisingadministering to a patient identified to be in need of prevention, ordelaying the onset, of non-APL AML or MDS a prophylactically effectiveamount of a RARA agonist therapy (e.g., tamibarotene) or apharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method for treating apatient for non-APL AML or MDS previously treated with a treatmentregimen comprising chemotherapy by administering to such a patient atherapeutically effective amount of a RARA agonist therapy (e.g.,tamibarotene) or a pharmaceutically acceptable salt thereof. In someembodiments, the present disclosure provides a method for treating apatient for non-APL AML or MDS where no standard therapies exist. Insome embodiments, the present disclosure provides a method for treatinga patient that is not suited for standard therapy.

In some embodiments, a patient may also have diseases associated withMDS, such as bone marrow failure, peripheral blood cytopenias andassociated complications of anemia, infection or hemorrhage. In someembodiments, a patient may have MDS that progresses to AML.

In some embodiments, a patient or patient population may not be (e.g.,may exclude) a patient who is or may be pregnant. In some embodiments, apatient or patient population may be monitored for one or more signs ofpregnancy, delivery, and/or lactation prior to and/or duringadministration of RARA agonist therapy. In some embodiments, RARAagonist therapy may be reduced, suspended, or terminated for a patientwho is determined to display one or more signs of pregnancy, delivery,and/or lactation.

In some embodiments, a patient or patient population may not be (e.g.,may exclude) a patient who has a previous history of hypersensitivity toan ingredient of tamibarotene. In some embodiments, a patient or patientpopulation may not be (e.g., may exclude) a patient who is receivingvitamin A formulations. In some embodiments, a patient or patientpopulation may not be (e.g., may exclude) a patient who hashypervitaminosis A.

In some embodiments, a patient or patient population may not be (e.g.,may exclude) an elderly patient. In some embodiments, a patient orpatient population may be or include one or more elderly patients. Insome embodiments, an elderly patient may be monitored more frequently todetect potential adverse events (including for example, low levels ofserum albumin and/or elevated concentrations of free drug in plasma,etc) as compared with one or more younger patients. In some embodiments,RARA agonist therapy may be reduced, suspended, and/or terminated for anelderly patient determined to display one or more signs of such anadverse event.

In some embodiments, a patient or patient population may not be (e.g.,may exclude) a pediatric patient. In some embodiments, a patient orpatient population may be or include one or more pediatric patients. Insome embodiments, a pediatric patient may be monitored more frequentlyto detect potential adverse events (including for example, increasedintracranial pressure, etc.) as compared with one or more olderpatients. In some embodiments, RARA agonist therapy may be reduced,suspended, and/or terminated for a pediatric patient determined todisplay one or more signs of such an adverse event.

In some embodiments, RARA agonist therapy in accordance with the presentdisclosure is reduced, suspended or terminated for a particular patientif and when the patient develops one or more adverse reactions such as,for example headache, rash, dry skin, eczema, exfoliative dermatitis,bone pain, joint pain, fever, increased leucocyte count, decreasedhaemoglobin, increased AST, increased ALT, increased LDH, increased ALP,increased TG, increased TC, follicitis, folliculitis, increased CRP, andcombinations thereof. Alternatively or additionally, in someembodiments, RARA agonist therapy in accordance with the presentdisclosure is reduced, suspended or terminated for a particular patientif and when the patient develops one or more adverse reactions such as,for example thrombosis (e.g., brain infarction, pulmonary infarction,arterial thrombosis, venous thrombosis, etc.), vasculitis, delirium,toxic epidermal necrosis (Lyell syndrome), erythema multiforme,increased intracranial pressure, and combinations thereof.

In some embodiments, the present invention provides use of a compound(e.g., tamibarotene) or a pharmaceutically acceptable salt thereof forthe manufacture of a medicament useful for treating, preventing, ordelaying the onset of non-APL AML, or MDS. In some embodiments, thepatient is suffering from cancer (e.g., non-APL AML or MDS). In someembodiments, the patient is suffering from cancer (e.g., non-APL AML orMDS) that is resistant to other therapies (e.g., chemotherapy). In someembodiments, the cancer is determined to have an IRF8 biomarker, whereinthe IRF8 biomarker is or comprises expression of one or more of elevatedIRF8 mRNA levels or a super enhancer associated with an IRF8 gene. Insome embodiments, the cancer is determined to express or more ofelevated RARA mRNA levels or a super enhancer associated with a RARAgene. In some embodiments, the cancer is determined not to express ormore of elevated RARA mRNA levels or a super enhancer associated with aRARA gene.

Dose Forms and Dosing Regimens

In general, each active agent (e.g., tamibarotene) for use in accordancewith the present invention is formulated, dosed, and administered in atherapeutically effective amount using pharmaceutical compositions anddosing regimens that are consistently with good medical practice andappropriate for the relevant agent(s) and subject. In principle,therapeutic compositions can be administered by any appropriate methodknown in the art, including, without limitation, oral, mucosal,by-inhalation, topical, buccal, nasal, rectal, or parenteral (e.g.,intravenous, infusion, intratumoral, intranodal, subcutaneous,intraperitoneal, intramuscular, intradermal, transdermal, or other kindsof administration). In some embodiments, a RARA agonist (e.g.,tamibarotene) will be administered orally.

In some embodiments, a dosing regimen for a particular active agent mayinvolve intermittent or continuous administration, for example toachieve a particular desired pharmacokinetic profile or other pattern ofexposure in one or more tissues or fluids of interest in the subjectreceiving therapy.

In some embodiments, different agents administered in combination may beadministered via different routes of delivery and/or according todifferent schedules. Alternatively or additionally, in some embodiments,one or more doses of a first active agent is administered substantiallysimultaneously with, and in some embodiments via a common route and/oras part of a single composition with, one or more other active agents.

Factors to be considered when optimizing routes and/or dosing schedulefor a given therapeutic regimen may include, for example, the particularindication being treated, the clinical condition of a subject (e.g.,age, overall health, prior therapy received and/or response thereto,etc.) the site of delivery of the agent, the nature of the agent, themode and/or route of administration of the agent, the presence orabsence of combination therapy, and other factors known to medicalpractitioners. For example, in the treatment of cancer, relevantfeatures of the indication being treated may include, among otherthings, one or more of cancer type, stage, location, etc.

In some embodiments, one or more features of a particular pharmaceuticalcomposition and/or of a utilized dosing regimen may be modified overtime (e.g., increasing or decreasing amount of active in any individualdose, increasing or decreasing time intervals between doses, etc.), forexample in order to optimize a desired therapeutic effect or response.

In general, type, amount, and frequency of dosing of active agents inaccordance with the present invention are governed by safety andefficacy requirements that apply when relevant agent(s) is/areadministered to a mammal, preferably a human. In general, such featuresof dosing are selected to provide a particular, and typicallydetectable, therapeutic response as compared with what is observedabsent therapy. In some embodiments, a RARA agonist (e.g., tamibarotene)will be administered continuously.

In context of the present invention, an exemplary desirable therapeuticresponse may involve, but is not limited to, inhibition of and/ordecreased tumor growth, tumor size, metastasis, one or more of thesymptoms and side effects that are associated with a tumor, as well asincreased apoptosis of tumor cells, therapeutically relevant decrease orincrease of one or more cell marker or circulating markers and the like.Such criteria can be readily assessed by any of a variety ofimmunological, cytological, and other methods that are disclosed in theliterature.

In some embodiments, it may be desirable to tailor dosing regimens, andparticularly to design sequential dosing regimens, based on timingand/or threshold expression levels of inducible markers, whether forparticular types of tumors, particular tumors, particular patientpopulations (e.g., carrying genetic markers), and/or particularpatients. In some such embodiments, therapeutic dosing regimens may becombined with or adjusted in light of detection methods that assessexpression of one or more inducible markers prior to and/or duringtherapy.

In some embodiments, a RARA agonist (e.g., tamibarotene) therapy regimencomprises at least one (or includes or consists of exactly one) dose ofabout 1 mg/m², 2 mg/m², 3 mg/m², 4 mg/m², 5 mg/m², 6 mg/m², 7 mg/m², 8mg/m², 9 mg/m², 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15mg/m², 16 mg/m², or a dose between any two of these values oftamibarotene. In some embodiments, a tamibarotene therapy regimencomprises a dose of 6 mg/m². In some embodiments, a tamibarotene therapyregimen comprises a dose of 4 mg/m². In some embodiments, a tamibarotenetherapy regimen comprises a dose of 2 mg/m². In some embodiments, atamibarotene therapy regimen comprises a dose of 1 mg/m².

In some embodiments, a RARA agonist (e.g., tamibarotene) therapy regimencomprises a plurality of doses of a tamibarotene composition. In somesuch embodiments, a tamibarotene therapy regimen comprises, for example2, 5, 10, 20, 30, 60, 90, 180, 365 doses or a number of doses betweenany two of these values and/or comprises a repeated pattern of doses(e.g., at least one cycle of two daily doses, which cycle may berepeated, optionally with a period of alternative administration, oroptionally no administration, separating different cycles). In someembodiments, a tamibarotene therapy regimen is administered twice a day.In some embodiments, a tamibarotene therapy regimen is administered oncea day. In some embodiments, a tamibarotene therapy regimen comprises atotal dose of 6 mg/m², divided as twice daily oral dosing.

In some embodiments, a RARA agonist (e.g., tamibarotene) therapy regimenmay be administered to a subject or population of patients known to haveconsumed, or not consumed, some amount of food before, during or afterthe administration. The terms “before administration” and “afteradministration” with respect to food intake may refer to a period oftime of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22,24, 30, 42, or 72 hours, or longer, before or after the administration.In some embodiments, the term “administering . . . with regard to foodintake” implies that the subject or population of patients consumes foodbefore the administration (e.g., fed state). In some embodiments, theterm “administering . . . with regard to food intake” implies that thesubject or population of patients consumes food after theadministration. In some embodiments, the term “administering . . . withregard to food intake” implies that the subject or population ofpatients consumes food during the administration. Alternatively, in someembodiments, the term “administering . . . with regard to food intake”means the subject or population of patients is in a fasted state duringadministration.

In some embodiments, food intake includes high fat foods or a high fatdiet. In some embodiments, a RARA agonist (e.g., tamibarotene) therapyregimen is administered to a subject in a fasted state. In someembodiments, a RARA agonist (e.g., tamibarotene) therapy regimen isadministered to a subject in a fed state.

Formulations

A pharmaceutical composition, as used herein, refers to a mixture of acompound, such as tamibarotene, with other chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, and/or excipients. The pharmaceutical compositionfacilitates administration of the compound to an organism.Pharmaceutical compositions containing a compound may be administered intherapeutically effective amounts by any conventional form and routeknown in the art including, but not limited to: intravenous, oral,rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal,vaginal, otic, nasal, and topical administration.

One may administer the compound in a local rather than systemic manner,for example, via injection of the compound directly into an organ, oftenin a depot or sustained release formulation. Furthermore, one mayadminister pharmaceutical composition containing a compound in atargeted drug delivery system, for example, in a liposome coated withorgan-specific antibody. The liposomes will be targeted to and taken upselectively by the organ. In addition, the pharmaceutical compositioncontaining a compound may be provided in the form of a rapid releaseformulation, in the form of an extended release formulation, or in theform of an intermediate release formulation. In some embodiments, theextended release formulation releases the compound for over 1 hour, over2 hours, over 3 hours, over 4 hours, over 6 hours, over 12 hours, over24 hours, or more. In some embodiments, the extended release formulationreleases the compound at a steady rate for over 1 hour, over 2 hours,over 3 hours, over 4 hours, over 6 hours, over 12 hours, over 24 hours,or more.

For oral administration, a compound can be formulated readily bycombining the active compounds with pharmaceutically acceptable carriersor excipients well known in the art. Such carriers permit the compoundsdescribed herein to be formulated as tablets, powders, pills, dragees,capsules, liquids, gels, syrups, elixirs, slurries, suspensions and thelike, for oral ingestion by a subject to be treated. Generally,excipients such as fillers, disintegrants, glidants, surfactants,recrystallization inhibitors, lubricants, pigments, binders, flavoringagents, and so forth can be used for customary purposes and in typicalamounts without affecting the properties of the compositions. In someembodiments, the excipient is one or more of lactose hydrate, cornstarch, hydroxypropyl cellulose and/or magnesium stearate. In someembodiments, tamibarotene may be formulated with one or more of lactosehydrate, corn starch, hydroxypropyl cellulose and/or magnesium stearate.

The identification of acceptable formulations of tamibarotene can beachieved by various methods known in the art, for example as describedin US 20100048708, which is incorporated herein by reference.

Combination Therapy

Those of ordinary skill in the art, reading the present disclosure, willreadily appreciate that a RARA agonist (e.g., tamibarotene), asdescribed herein, may in certain embodiments be combined with otheranti-cancer therapies, including for example administration ofchemotherapeutic agents, other immunomodulatory agents, radiationtherapy, high-frequency ultrasound therapy, surgery, FDA approvedtherapies for treatment of cancer, etc.

In some embodiments, a RARA agonist is utilized in combination with oneor more other therapeutic agents or modalities. In some embodiments, theone or more other therapeutic agents or modalities is also ananti-cancer agent or modality; in some embodiments the combination showsa synergistic effect in treating cancer.

Known compounds or treatments that show therapeutic efficacy in treatingcancer may include, for example, one or more alkylating agents,anti-metabolites, anti-microtubule agents, topoisomerase inhibitors,cytotoxic antibiotics, angiogenesis inhibitors, immunomodulators,vaccines, cell-based therapies, organ transplantation, radiationtherapy, surgery, etc.

In some embodiments, a RARA agonist (and/or other therapy with which itis combined) may be combined with one or more palliative (e.g., painrelieving, anti-nausea, anti-emesis, etc.) therapies, particularly whenrelieves one or more symptoms known to be associated with the relevantcancer, or with another disease, disorder or condition to which aparticular cancer patient is susceptible or from which the particularcancer patient is suffering.

In some embodiments, agents used in combination are administeredaccording to a dosing regimen for which they are approved for individualuse. In some embodiments, however, combination with a RARA agonist(e.g., tamibarotene) permits another agent to be administered accordingto a dosing regimen that involves one or more lower and/or less frequentdoses, and/or a reduced number of cycles as compared with that utilizedwhen the agent is administered without a RARA agonist (e.g.,tamibarotene). Alternatively or additionally, in some embodiments, anappropriate dosing regimen involves higher and/or more frequent doses,and/or an increased number of cycles as compared with that utilized whenthe agent is administered without a RARA agonist (e.g., tamibarotene).

In some embodiments, one or more doses of agents administered incombination are administered at the same time; in some such embodiments,agents may be administered in the same composition. More commonly,however, agents are administered in different compositions and/or atdifferent times. In some embodiments, tamibarotene is administeredsequentially and/or concurrently with other therapeutic agents (e.g.,chemotherapy).

In some embodiments, the combination therapies disclosed herein are onlyadministered if a subject has a RARA mRNA level equal to or above athreshold value. In some embodiments, the combination therapiesdisclosed herein are only administered if a subject has an IRF8 mRNAlevel equal to or above a threshold value. In some embodiments, thecombination therapies disclosed herein are only administered to asubject that has both a RARA mRNA level equal to or above a thresholdvalue and an IRF8 mRNA level equal to or above a threshold value. Insome aspects of any of these embodiments, the subject is suffering fromnon-APL AML.

In some embodiments, the therapeutic agent to be combined with a RARAagonist (e.g. tamibarotene) is selected from a DNA methyltransferaseinhibitor, a DNA synthase inhibitor, a topoisomerase inhibitor, a FLT3inhibitor, a folate inhibitor, a BRD4 inhibitor, a Zn fingertranscription factor inhibitor, a GCR inhibitor, a CDK7 inhibitor, anHDAC inhibitor, a JMJD3/JARID1B inhibitor, or an EZH2 inhibitor. Inother specific aspects, that second agent is selected from a LSD1inhibitor, a proteasome inhibitor, a DNA damage repair inhibitor, a PARPinhibitor, a mTOR inhibitor, a DOT1L inhibitor, a tubulin inhibitor, aPLK inhibitor, or an Aurora kinase inhibitor.

In some embodiments, a RARA agonist (e.g., tamibarotene) can beadministered with decitabine, azacitidine, ara-C, daunorubicin,idarubicin, arsenic trioxide and/or flt3 inhibitors. In someembodiments, a RARA agonist (e.g., tamibarotene) can be administeredwith IDH inhibitors, BRD4 inhibitors (e.g., JQ1), HDAC inhibitors (e.g.,SAHA and MC1568), HMT inhibitors (e.g., EPZ6438, UNC0638, SGC707,EPZ5676, UNC037 and PFI-2) and/or KDM inhibitors (e.g., GSKJ4, RN-1 andGSK-LSD1).

In some embodiments, the subject is suffering from AML and tamibaroteneis administered in combination with a second agent selected fromazacytidine, arsenic trioxide, midostaurin (only in those AML subjectscharacterized by high FLT3 mRNA levels), cytarabine, daunorubicin,methotrexate, idarubicin, sorafenib (only in those AML subjectscharacterized by high FLT3 mRNA levels), decitabine, quizartinib (onlyin those AML subjects characterized by high FLT3 mRNA levels), JQ1 (aBRD4 inhibitor), ATO, prednisone (only in those AML subjectscharacterized by high GCR mRNA levels), SAHA, and GSKJ4 (only in thoseAML subjects characterized by high JMJD3/JARID1B mRNA levels).

Kits

A kit comprising one or more reagents for detecting one or more IRF8biomarkers can be provided in a kit. In some instances, the kit includespackaged pharmaceutical compositions of the present invention comprisinga written insert or label with instructions to use a RARA agonist (e.g.,tamibarotene) in a subject suffering from a cancer and who has beendetermined to have a super enhancer associated with an IRF8 gene havinga strength, or ordinal rank equal to or above a threshold level, or anIRF8 mRNA level equal to or above a reference (e.g., threshold level).As described in detail above, the threshold level is determined in apopulation of samples from either subjects diagnosed as suffering fromthe same disease or cell lines or xenograft models of the same diseaseas that for which the pharmaceutical composition is indicated fortreatment. The instructions may be adhered or otherwise attached to avessel comprising a RARA agonist. Alternatively, the instructions andthe vessel comprising a RARA agonist will be separate from one another,but present together in a single kit, package, box, or other type ofcontainer.

The instructions in the kit will typically be mandated or recommended bya governmental agency approving the therapeutic use of a RARA agonist.The instructions may comprise specific methods of determining whether asuper enhancer is associated with an IRF8 gene, as well asquantification methods to determine whether an enhancer associated withan IRF8 gene is a super enhancer, quantification methods to determineIRF8 mRNA levels; and/or threshold levels of super enhancers or IRF8mRNA at which treatment with a packaged RARA agonist is recommendedand/or assumed therapeutically effective. In some aspects, theinstructions direct that the composition be administered to a subjectwhose IRF8 mRNA level falls in at least the 30^(th) percentile of apopulation whose IRF8 mRNA levels have been measured. In some aspects ofthese embodiments, a subject is identified as a RARA agonist responderif its IRF8 mRNA level prevalence rank is about 80%, 79%, 78%, 77%, 76%,75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%,61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%, 48%,47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%,33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%in a population whose IRF8 mRNA levels have been measured. In someaspects, the instructions direct that the composition be administered toa subject whose IRF8 mRNA level as measured by a specific assay

The instructions may optionally comprise dosing information, the typesof cancer for which treatment with a RARA agonist was approved,physicochemical information about a RARA agonist; pharmacokineticinformation about a RARA agonist, drug-drug interaction information. Insome embodiments, the instructions direct that the composition beadministered to a subject diagnosed as suffering from AML. In someembodiments, the instructions direct that the composition beadministered to a subject diagnosed as suffering from non-APL AML. Insome aspects, the instructions direct that the composition beadministered to a subject diagnosed as suffering from MDS. In someaspects, the pharmaceutical composition comprises tamibarotene.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The synthetic andbiological examples described in this application are offered toillustrate the compounds, pharmaceutical compositions, and methodsprovided herein and are not to be construed in any way as limiting theirscope.

Example 1: IRF8 mRNA Levels in Non-APL AML Cell Lines Correlate withResponsiveness to a RARA Agonist

We previously tested several AML cell lines for sensitivity totamibarotene and demonstrated that sensitivity correlated very well witheach of RARA super enhancer strength, RARA super enhancer strengthordinal and RARA mRNA level. This was done as described below.

On the day of the experiment, cells were homogenized using Accumax (EMDMillipore), counted, and adjusted to 60,000 cells/mL in appropriategrowth media. Using a Biotek EL406, 50 μl of cells were distributed intowhite (ATPlite) or black (CyQuant) 384-well plates (Thermo). Cells werereturned to 37° C. incubator to allow adhesion. After three hours,compounds were added to plates using a 20 nl 384-well pin transfermanifold on a Janus workstation. Stocks were arrayed in 10 pointquadruplicate dose response in DMSO stock in 384-well compound plates.After addition of compound, plates were incubated for five or ten daysin a 37° C. incubator.

Cell viability was read out using ATPlite (Perkin Elmer) or CyQuant(Life Technologies). For ATPlite, plates were removed from the incubatorand brought to room temperature prior to use. Lyophilized powder ofATPlite reagent was resuspended in lysis buffer and diluted 1:2 withdistilled water. 25 μL of this solution was added to each well using theBiotek liquid handler. Plates were incubated for 15 min at roomtemperature before the luminescence signal was read on an Envision PlateReader (Perkin Elmer). For CyQuant, reagents were mixed as permanufacturer's instructions in PBS (Gibco). Reagent was added using amultichannel pipet and plates were replaced in incubator for 30 minutesprior to readout on an Envision Plate Reader (Perkin Elmer).

Data acquired as described was stored and grouped in Microsoft's Exceland analyzed using GraphPad Prism Software. Curve fits to calculate EC₅₀and E_(max) were done in GraphPad Prism version 6.0 using four parameter(Hill slope not assumed to be equal to 1) non-linear regressions withthe log 10 transformed data of the compound concentrations plottedagainst the percent viability of the cells when normalized to DMSO onlytreated wells included on the plate. Edge wells were excluded.

We used an Affymetrix GeneChip® PrimeView™ Human Gene Expression Arrayto initially examine seven of these AML cell lines (four sensitive totamibarotene—NOMO-1, OCI-AML3, MV-4-11, and Sig-M5; and threeinsensitive—KG1a, OCI-M1 and Kasumi-1) for other mRNAs that might bespecifically elevated in the tamibarotene sensitive cell lines andidentified IRF8 mRNA as a potential candidate. We then quantified IRF8mRNA levels in each of these seven AML cell lines previously, as well asseveral other AML cell lines tested for sensitivity to tamibarotene byperforming RNA-seq analysis as set forth below. The results for thefirst seven cell lines are shown in FIG. 1. Interestingly, NOMO-1 didnot have a high RARA mRNA level, but was responsive to tamibarotene. Thefact that NOMO-1 had elevated IRF8 mRNA levels helped clarify thisseeming inconsistency and further validated the use of IRF8 mRNA levelsto predict responsiveness to tamibarotene.

RNA preparation: Cell suspension was transferred to microcentrifugetubes and washed with 1 mL of PBS. Cell pellets were re-suspended in 200μL of TRIzol. 20 μL of miRNA Homogenate Additive from the Ambion miRVanamiRNA Isolation Kit (AM1561) was added, mixed, and incubated on ice for10 minutes. 20 μL of bromochloropropane was added, mixed, incubated atroom temperature for 5 minutes, and centrifuged at 12,000×g for 10minutes at 4° C. 62 μL of the aqueous phase was added to 78 μL ofethanol and transferred to a filter column. The isolation was continuedaccording to Ambion's Total RNA Isolation protocol. Sample was testedfor quality control on a bioanalyzer and then sent to the WhiteheadSequencing Core, (Cambridge, Mass.) for sequencing.

RNA-seq data processing: Reads were aligned to the HG19 transcriptomeusing rsem v1.2.21 software (rsem-calculate-expression; parameters=-p4--samtools-sort-mem 3G--ci-memory 3072--bowtie-chunkmbs1024--quiet--output-genome-bam--bowtie2--bowtie2-path/data/devtools/bowtie2-2.0.5--strand-specific)and then mRNA quantification was done using the same rsem suite(rsem-parse-alignments, rsem-build-read-index, rsem-run-em) and reportedin transcripts per million (TPM). All protein coding genes were thenextracted for each sample and their scores were quantile normalized.

We then compared sensitivity to tamibarotene to IRF8 mRNA levels asshown in FIG. 2 and Table 1.

TABLE 1 AML cell line IRF8 mRNA levels and tamibaroteneanti-proliferative potency Tamibarotene anti- IRF8 mRNA proliferativepotency Cell Line (TPM) (EC_(50,) nM) EOL-1 484.38 0.89 Kasumi-116.34 >50000 KG-1a 1.84 >50000 PL21 190.02 1.41 MV4-11 699.41 0.17 HL60*6.73 1.64 OCI-AML3 739.29 0.34 OCI-AML2 451.94 0.34 Nomo-1 242.19 0.48OCI-M1 1.02 >50000 HEL 1.00 >50000 *HL60 is an APL cell line.

As can be seen from the above table, all tamibarotene-responsive celllines, except for HL60, had an IRF8 mRNA level of greater than 190 TPM(log 2(7.57)) in the assay, while non-responsive cell lines all had anIRF8 mRNA level of less than 16.5 TPM (log 2(4.03)). The responsivenessof HL60 to tamibarotene without a concomitant high level of IRF8 mRNA(6.73 TPM) suggests that correlation between IRF8 mRNA level andtamibarotene sensitivity may not hold for APL and thus may be bettersuited to stratify subjects suffering from non-APL AML. FIG. 2 removesthe data point for HL60.

IRF8 mRNA levels were determined for a large number of different typesof samples—normal blood cells, AML cell lines, primary AML patientsamples and AML PDXs. Data obtained were plotted in rank order, and theresults are presented graphically in FIG. 14. As can be seen, FIG. 14does not show any correlation between IRF8 mRNA levels and presence ofdisease; and IRF8 levels appear to be distributed in a reasonablysimilar manner in normal cells as compared with diseased cells, celllines and PDXs.

Example 2: Determination of IRF8 mRNA Threshold Values for RARA AgonistTreatment

The AML cell line results suggest a cutoff value of between 15.5 and 190TPM (i.e., between log 2(4.03) and log 2(7.57) in the RNA-Seq assay. Wechose a population of AML patient samples (kindly provided by StanfordUniversity) in order to examine the distribution of IRF mRNA levels andto determine prevalence cutoffs based on the cutoff values. We added tothat population AML cell lines and then generated a rank-ordered graph.FIG. 3 shows that rank-ordered distribution of IRF8 mRNA levels in thecombined patient sample/AML cell line population. We determined that aprevalence cutoff of 25% corresponded to an IRF mRNA value ofapproximately log 2(7).

Example 3: Correlation of IRF8 mRNA and RARA mRNA Levels

We next compared IRF8 and RARA mRNA levels in AML cell lines and patientpopulation to determine correlation. FIG. 4 shows that some cell linesthat responded to tamibarotene have relatively low RARA mRNA, but a highlevel of IRF8 mRNA. FIG. 5 shows that a subset of patients, too,demonstrates high IRF8 mRNA levels, but relatively low RARA mRNA levelsand vice versa. This supports the idea that measuring both IRF8 and RARAmRNA in a patient and selecting that patient for treatment with a RARAagonist, such as tamibarotene, if either mRNA level is above a thresholdvalue may optimize the treatable patient population.

Example 4: A Super Enhancer Associated with IRF8 Correlates withResponsiveness to RARA Agonist Treatment

We next examined IRF8 enhancer strength in several AML cell lines andpatient samples as follows.

Cell Fixation: For cells in suspension, typically a 1/10 volume of fresh11% formaldehyde solution was added to cell suspension, mixed and themixture was allowed to sit at room temperature (RT) for 8 min. Then 1/20volume of 2.5 M glycine or ½ volume of 1 M Tris pH 7.5 was added toquench formaldehyde and incubated for at least 1 min. Cells were rinsed3 times with 20-50 mL cold 1× phosphate-buffered saline (PBS),centrifuged for 5 min at 1250×g to pellet the cells before and aftereach wash. Cells were then transferred to 15 mL conical tubes andcentrifuged at 1250×g for 5 minutes at 4° C. The supernatant wasremoved, residual liquid was removed by dabbing with a Kimwipe, and thenthe pelleted cells were flash frozen in liquid nitrogen and stored at−80° C.

Bead Preparation: Approximately 60 μL of Dynabeads® Protein G per 2 mLimmunoprecipitate (Invitrogen) were used. Beads were washed 3 times for5 minutes each with 1.0 mL blocking buffer (0.5% BSA w/v in PBS) in a1.5-mL Eppendorf tube. A magnet (Invitrogen) was used to collect thebeads (and allowed magnet binding for at least 1 full minute) after eachwash and the supernatant was then aspirated. The washed beads werere-suspended in 250 μL blocking buffer to which 6 μg of antibody wasadded and the mixture was allowed to incubate with end-over-end mixingovernight (minimum 6 hours). The antibody-bound beads were washed 3times for 5 min each with 1 mL blocking buffer and re-suspended inblocking buffer (60 μL per IP). These last washes and resuspensions weredone once the cells were sonicated (see 9.1.1.3) and just prior toovernight immunoprecipitation.

Cell Lysis: Protease inhibitors at 1× (Complete, Roche; prepared bydissolving one tablet in 1 mL H₂O for 50× solution and stored inaliquots at −20° C.) were added to all lysis buffers before use. Eachtube of cells (approximately 5×10⁷ cells) was re-suspended in 5-10 mL oflysis buffer 1 (LB1; 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40,0.25% Triton X-100) and rocked at 4° C. for 10 minutes. The cells werecentrifuged at 1250×g×5 min in tabletop centrifuge at 4° C. and thesupernatant aspirated off. The cells were re-suspended in 5 mL LysisBuffer 2 (LB2; 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8) andincubated end-over-end at 4° C. for 10 minutes. The cells were againpelleted at 1250×g for 5 min in tabletop centrifuge at 4° C. and washedin 2-5 mL Covaris sonication buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.1%SDS). The pellet was centrifuged at 1250×g for 5 min in tabletopcentrifuge at 4° C. The cells were pelleted at 1250×g for 5 min in atabletop centrifuge at 4° C. and re-suspended at a concentration of20-50 million cells/1 mL of Covaris sonication buffer.

Chromatin Immunoprecipitation: Fifty μL of antibody-conjugated beadsprepared as described above was added to the cleared cellular extract(as described above in Cell Lysis) solution in 1.5 ml tubes and rockedovernight at 4° C. (minimum 8 hours) to immunoprecipitate DNA-proteincomplexes.

Wash, elution, and cross-link reversal: All buffers used in these stepswere ice cold. A magnetic stand was used to precipitate magnetic beads,washed 3 times, 5 minutes each, with gentle end-over-end mixing with 1mL Wash Buffer 1 (50 mM HEPES pH 7.5; 140 mM NaCl; 1 mM EDTA; 1 mM EGTA;0.75% Triton-X; 0.1% SDS; 0.05% DOC); washed once for 5 minutes with 1mL Wash Buffer 2 (50 mM HEPES pH 7.5; 500 mM NaCl; 1 mM EDTA; 1 mM EGTA;0.75% Triton-X; 0.1% SDS; 0.05% DOC); and once for 5 minutes with 1 mLWash Buffer 3 (10 mM Tris pH 8.0; 1 mM EDTA; 50 mM NaCl). All residualwash buffer was aspirated and the beads were centrifuged gently at1250×g for 1 min; the tubes were replaced onto the magnet and all tracesof buffer were removed. Elution buffer at a volume of 210 was added (50mM Tris pH 8; 10 mM EDTA; 1% SDS) and eluted at 65° C. for 60 min withbrief vortexing to re-suspend beads every 15 min. The beads wereseparated from the supernatant using the magnet and 200 μL ofsupernatant was removed and placed in a clean tube for reversecross-linking. Both IP and whole cell extract fractions were reversex-linked overnight at 65° C. (minimum 8 hours, but maximum 18 hours).Heating was then used to separately reverse cross-linked both the samplefor immunoprecipitation and the whole cell extract fractions byincubating overnight at 65° C. (minimum 8 hours, but maximum 18 hours).The heating facilitated the hydrolysis of the formaldehyde cross-links.

Cleanup and purification of DNA: Tris-EDTA buffer (50 mM Tris pH 8; 1 mMEDTA) and 2.7 μL of 30 mg/ml RNaseA (0.2 mg/mL final concentration) at avolume of 200 was added to each sample, mixed and incubated at 37° C.for 2 hours. Then 5 μL of calcium chloride solution (300 mM CaCl₂ in 10mM Tris pH 8.0) was added to each sample along with 4 of 20 mg/mlproteinase K (0.2 mg/mL final concentration), mixed and incubated at 55°C. for 60 minutes. Then 400 μL of phenol:chloroform:isoamyl alcohol at25:24:1 ratio (Sigma Aldrich #P3803) was added to each tube, mixed on avortex mixer on low setting (5/10) and inverted each tube to mixfurther.

A PhaseLock Gel™ tube (Qiagen, 3 Prime) was prepared for each sample bycentrifuging the tube at room temperature for 30 seconds at 10,000 RPM.Next, the sample DNA in phenol:chloroform:isoamyl alcohol was added tothe PhaseLock Gel™ tube and centrifuged at 12,000-16,000×g for 2 minutesat room temperature. Then the aqueous solution was transferred to a new1.6 mL tube (top fraction), added 20 μL of 5 M NaCl, and 1.5 μL of 20μg/μL glycogen (30 μg total), then added 1 mL of EtOH, and mixed byvortex or inversions. The sample was then incubated at −20° C. overnight(6-16 hours). The mixture was centrifuged at 20,000×g for 20 minutes at4° C. to pellet the DNA, the supernatant was removed with 1 mL pipettetip, washed the pellets in 800 μL of 80% EtOH, centrifuged at 20,000×gfor 20 minutes at 4° C., and removed the supernatant with 1 mL pipettetip. The sample was centrifuged again for 1 min at 20,000×g, supernatantremoved, and the pellet allowed to air dry for 5-20 minutes. The pelletsshould not have a halo of water around them and should be glassy orflaky dry. The pellet was then dissolved in 60 μL of water, using 50 μLfor sequencing.

ChIP-seq data processing: Reads for both the ChIP-seq IP and IN werealigned to the HG19 genome using Bowtie2 v.2.0.5 software (parameters=-p4-sensitive). This resulted in genome-wide BAM files summarizing thealignment of both the IP and IN sequencing experiments.

Creating a universal IRF8 enhancer score dataset: A universal IRF8enhancer score dataset was generated that could apply in all downstreamanalyses. Peaks observed genome-wide in the aligned H3K27Ac read datawith MACS v1.4 using the aligned IP BAM were designated as the ChIP-seqforeground data and the aligned IN BAM as the control background data. Astringent p value cutoff of 10⁻⁹ was used, but otherwise used thedefault parameters. These peaks were then merged if they had ≥12,500base pairs between them in the human reference genome. This set of peaksis referred to as the ROSE peaks, and the rank of the highest-scoringROSE peak overlapping the IRF8 transcript for a given sample is recordedas “IRF8 ROSE Rank” for that sample.

The set of ROSE peaks was then filtered for “blacklist regions” asdefined by ENCODE(https://sites.google.com/site/anshulkundaje/projects/blacklists) andENCODE Project Consortium (2012) to remove ChIP-seq artifacts.

The filtered set of ROSE peaks from the primary patient samples werethen merged into a universal H3K27Ac enrichment map by taking the unionof the coordinates of each peak from a given sample with all of thepeaks that overlapped it from the other samples. This generated auniversal map of H3K27Ac enrichment. Then each enriched region wasquantified within this universal map within each sample (including celllines) by, for a given region, summing the number of IP reads thatmapped within the region and dividing by the number of reads mapping inthe full experiment multiplied by a million (“reads per million”, orRPM). A similar RPM score for the IN reads was calculated. The IN RPMwas subtracted from the IP RPM to achieve the overall score for a givenregion within the universal map for a given sample. A negative binomialdistribution was fit to the scores for a given sample using the fitdistrfunction in the R MASS library v7.3.45. The tail of the distribution waslocated as the point where the cumulative distribution function of thisnegative binomial crossed 0.99 (equivalent to a p value of 0.01). Theoverall scores for all of the sample's regions by this point weredivided, so that any region of enrichment that scored in the bottom 99%of the fitted negative binomial distribution scored below 1 (deemed a“typical enhancer”) and any region that scored above the 99% mark scoresabove 1 (deemed a “super-enhancer”). These scores are termed the“RECOMB” scores for each sample against a universal map. Each sample'sRECOMB scores was then normalized against all other samples usingquantile normalization, and with the floor set at 0.

Visualization of ChIP-seq data: The genome-wide localization of H3K27Acwas visualized using the Integrative Genomics Viewer (IGV) version2.3.60 after converting the BAM files to IGV formatted t files usingMACS2 to create a pileup (extsize 200) and the igvtools v2.3.9software's igvtools to TDF command. The Y-axis of each track is set tobegin just above the level of noise (0.25) and end at a level thatapproximates half the level required to view the full height of the peakover a control region centered at the MALAT1 gene.

TABLE 2 AML cell line IRF8 enhancer strength, mRNA level andtamibarotene anti-proliferative potency Tamibarotene anti- IRF8 enhancerIRF8 mRNA proliferative potency Cell Line (RECOMB) (log₂) (EC_(50,) nM)EOL-1 0.77 8.96 0.89 Kasumi-1 1.22 4.06 >50000 KG-1a 0.00 0.68 >50000PL21 3.10 7.69 1.41 MV4-11 2.44 9.48 0.17 HL60* 0.17 2.63 1.64 OCI-AML31.72 9.65 0.34 OCI-AML2 1.36 8.88 0.34 Nomo-1 1.58 8.04 0.48 OCI-M1 0.000.04 >50000 HEL 0.00 0.00 >50000 Sig-M5 1.53 8.53 0.46 THP-1 1.87 8.730.95 *HL60 is an APL cell line.

The data above demonstrates that an IRF8 RECOMB enhancer score of ≥1.0(a RECOMB score of ≥1.0 defines a super enhancer) correlates well withresponsiveness to tamibarotene. Excluding the HL60 APL cell line, thatcutoff value produced 1 false positive (Kasumi-1) and one false negative(EOL-1) out of twelve non-APL AML cell lines tested. Raising the cutoffto a RECOMB score of ≥1.25 would eliminate the false positive, whilelowering the cutoff to a RECOMB score of ≥0.75 would eliminate the falsenegative. This data is also presented in graph form in FIG. 6. A similarcorrelation between IRF8 mRNA level and responsiveness to tamibarotenewas also observed, with cell lines having a IRF8 mRNA TPM(log₂) value ofgreater than 4.25 all demonstrating tamibarotene sensitivity.

We then applied enhancer profiling by ChIP-seq to a subset of AMLpatient samples. The IRF8 locus enhancer strength varied widely amongthe 66 AML patient samples, with 21% (14/66) of the patients having a SEindicated by RECOMB scores surpassing 1.0 (FIG. 7). Most patient samplesexhibited minimal enhancer activity, including the lowest 14% (9/66)which had no quantifiable IRF8 enhancer.

Example 5: Correlation of IRF8 mRNA and IRF8 Enhancer Strength

Quantification of IRF8 enhancer and correlation of ChIP-seq and RNA-seqdata: The quantile-normalized RECOMB score was used across all patientsfor the region called as an enhancer in the universal map thatoverlapped IRF8: chr16:85862582-85990086. This was correlated with thequantile-normalized TPM expression estimates for the full IRF8 genemodel from RSEM using Spearman correlation. Only patients with bothRNA-seq and ChIP-seq were used. The same analysis was performed in celllines, but excluded APL cell lines.

To enable proxy estimation of IRF8 SE by IRF8 mRNA measurement, thecorrelation between the two was examined in the same AML patient cohort.The IRF8 mRNA measured by RNA-seq was compared with the IRF8 locusenhancer measure by RECOMB score for the H3K27ac (FIG. 8). IRF8 mRNAlevels also varied widely among samples in this cohort and the IRF8 mRNAlevels were highly correlated with IRF8 enhancer strength (Spearman Rhocorrelation estimate ˜0.0.81, p-value of 2.2×10¹²).

We also profiled the value of IRF8 enhancer strength and IRF8 mRNAlevels in 26 AML cell lines. Several of these cell lines were testedpreviously for antiproliferative sensitivity to tamibarotene (see Table2). As observed in AML patient samples, AML cell lines exhibited a broaddistribution of IRF8 enhancer strengths (FIG. 9). IRF8 enhancer strengthand IRF8 mRNA levels also varied widely in 26 AML cell lines, 9 (34%) ofwhich had IRF8 RECOMB values of ≥1.0.

As with the ANIL patient samples, the AML cell lines exhibited a strongcorrelation of IRF8 mRNA levels with the IRF8 enhancer strength (FIG.10; Spearman Rho correlation estimate ˜0.0.82, p-value of 2×10⁻⁶), thussupporting IRF8 mRNA as a proxy measure of IRF8 enhancer strength.

Example 6: Response of PDX Models to Tamibarotene and Correlation withIRF8 mRNA Levels

Different AML patient sample (AM8096, AM5512, AM7577 and AM7440)-derivedxenograft models in BALB/c nude immunocompromised mice are prepared byCrown Biosciences (Beijing, China) essentially as follows.

Approximately 2×10⁶ cells from each patient sample are suspended in 100μL PBS and injected into separate mice (n=3 for each different patientsample and for the control) by IV tail injection. For AM5512, AM7577 andAM7440 xenografts, tumor burden is considered high enough to starttreatment when the concentration of human CD45⁺ cells reaches ˜1-5% inthe animal's peripheral blood. Human CD45⁺ cells are detected in mouseblood (obtained via eye bleeds) using a fluorescence activated cellsorter and FITC anti-human CD45 (Biolegend, Cat #304037). For AM8096xenografts, treatment is begun 40 days after injection of cells.

Tamibarotene is administered orally in pH 8 adjusted PBS, 1% DMSO on adaily schedule at a final dose of 6 mg per kg body weight in a 10 ml/kgvolume. Mice in the vehicle arm are given the same schedule, volume, andformulation, but lacking tamibarotene. Human CD45⁺ cell levels inperipheral blood from the treated animals and control animals aremeasured once a week.

AM5512 and AM8096 xenografts show a significant reduction in the total %of CD45⁺ cells, as well as in the % of CD45⁺ cells in blood, bone marrowand spleen, when treated with tamibarotene as compared to the vehiclecontrol after 35 days of treatment (FIG. 11). On the other hand, AM7577and AM7440 show no significant reduction in tumor volume between thetamibarotene treated and vehicle treated animals either overall or inany of blood, bone marrow or spleen (FIG. 12).

We then measured both the IRF8 and RARA mRNA levels in each of the fourpatient samples used in the xenograft study (FIG. 13). The twonon-responders in the xenograft study, AM7577 and AM7440, have IRF8 mRNAlevels that fall well below 100 TPM in the assay. One of the responders,AM8096, has an IRF8 mRNA level that is above 350 TPM. The otherresponder, AM5512, has a very low level of IRF8 mRNA. Interestingly,these four samples showed a similar pattern of RARA mRNA level, withAM7577 and AM7440 having RARA mRNA levels below any determinedprevalence cutoff (e.g., a 36% prevalence cutoff); AM8096 beingsignificantly above that prevalence cutoff and AM5512 also being belowthe prevalence cutoff, but having significantly higher RARA mRNA thaneither of the AM7577 or AM7440 non-responders.

Example 7: Obtaining and Preparing Patient Samples for Determining IRF8mRNA Levels and ChIP Sequencing

Blood (8 mL) was drawn from non-APL AML patients and collected into an 8mL BD Vacutainer CPT Sodium Citrate tube. Following blood collection,the tube was gently hand inverted 8-10 times to ensure adequate missingof the anticoagulant. The tube was stored upright at room temperatureprior to centrifugation which was performed within two hours ofcollection. The blood sample was then centrifuged at room temperature(18-25° C.) for 20 minutes at 1500-1800 RCF (relative centrifugalforce). After centrifugation, the blood separated into layers. Thebottom layers below the gel plug were a red layer at the absolute bottom(red blood cells) and a thin gray layer above this (granulocytes anddensity solution). Directly above the gel plug was a clear layer ofdensity solution, then a white layer (mononuclear cells and platelets)and a yellowish layer (plasma) on top. The white layer (up to 1 mL involume) containing PBMCs was removed immediately after centrifugationwith a Pasteur pipette. If necessary, the PBMCs can be stored in acryovial containing 20% v/v BloodStor® freezing media (BioLifeSolutions) which is added dropwise followed by gentle hand inversion tomix.

The PBMC fraction obtained in the previous step (thawed if previouslyfrozen) was then treated simultaneously with human CD117 MicroBeads(Miltenyi Biotec) and human CD34 MicroBeads (Miltenyi Biotec), followingmanufacturer's directions for magnetic labeling and magnetic separationof labeled cells. Messenger RNA was then extracted from the isolatedCD34⁺/CD117⁺ cells and quantitated using qPCR as described above.

Example 8: Synergy Between Tamibarotene and Other Agents Correlates withRARA mRNA Levels

Using a Biotek EL406, 50 μL of cell media containing 20-60,000 cells/mlwas distributed into white 384-well Nunc plates (Thermo). Suspensioncells then received compound immediately while adherent cells lines weregiven one hour to reattach to the surface of the plate prior to compoundaddition. Tamibarotene and the second agents to be tested were dissolvedin DMSO and arrayed on 384 well compound storage plates (Greiner). Eachcompound plate received tamibarotene and one second agent each in 5different doses centered approximately on the EC₅₀ of the given compoundfor a given cell line, providing a total of 25 different dosecombinations of the two agents.

Compound arrays were distributed to assay plates using a 20 nl 384-wellpin transfer manifold on a Janus MDT workstation (Perkin Elmer). Eachplate contained 8 replicates of all 5 by 5 compound concentrations inaddition to five doses of each compound on its own in quadruplicate.After addition of compounds, cell plates were incubated for 5 days in a37° C. incubator. Cell viability was evaluated using ATPlite (PerkinElmer) following manufacturer protocols. Data was analyzed usingcommercially available CalcuSyn software and visualized using GraphPadPrism Software. Isobolograms plotting each of the 25 dose combination oftamibarotene and the second agents were generated and analyzed for thepresence of synergy. In the isobolograms, the straight line connectingthe abscissa and the ordinate values of 1.0 represents growthinhibitions that were additive for the combination of the two compounds.Plots that fall below the straight line represented synergistic growthinhibitions, with plots that fall below that line and one connecting theabscissa and the ordinate values of 0.75 represent mild synergy. Plotsthat fall between a line connecting the abscissa and the ordinate valuesof 0.75 and a line connecting the abscissa and the ordinate values of0.25 represent moderate synergy. Plots that fall below a line connectingthe abscissa and the ordinate values of 0.25 represent strong synergy.Data points that are outside the maxima in each isobologram areindicated by the number of asterisks in the upper right hand corner ofthe isobologram and represent data points of no synergy.

We tested azacytidine, arsenic trioxide, midostaurin, cytarabine,daunorubicin, methotrexate, idarubicin, sorafenib, decitabine,quizartinib, ABT199 (a BCL2 inhibitor), JQ1 (a BRD4 inhibitor), ATO,prednisone, SAHA, GSKJ4 (a JMID3/JARID1B inhibitor) and EPZ6438 (an EZH2inhibitor) as second agents in these assays against various AML celllines. FIGS. 15-21 depict isobolograms for various second agents incombination with tamibarotene in different cell lines.

For combinations of azacytidine and tamibarotene, moderate to strongsynergy was observed for Sig-M5; moderate synergy was observed for KG-1aand NOMO-1; and mild-to-moderate synergy was observed for MV-4-11 (seeFIG. 15). No synergy was observed for Kasumi-1 or OCI-M1 (data notshown).

For combinations of arsenic trioxide and tamibarotene, strong synergywas observed for Sig-M5 and MV411; and moderate synergy was observed forNOMO-1 (see FIG. 16). None-to-mild synergy was observed for Kasumi-1,and no synergy was observed OCI-M1 (data not shown).

For combinations of cytarabine (Ara-C) and tamibarotene, some moderatesynergy was observed for KG-1a and OCI-M1, but no synergy was observedfor HL-60 (see FIG. 17). For MV-411 the large number of data pointsoutside the maxima (7 of 25) and the large number that showed strongsynergy makes interpretation difficult.

For combinations of daunorubicin and tamibarotene, strong synergy wasobserved for Kasumi-1 and NOMO-1; and moderate synergy was observed forSig-M5 and MV-4-11 (see FIG. 18). No synergy was observed OCI-M1 (datanot shown).

For combinations of methotrexate and tamibarotene, moderate synergy wasobserved for NOMO-1, Sig-M5 and MV-4-11 (see FIG. 19). None-to-mildsynergy was observed for Kasumi-1, and no synergy was observed forOCI-M1 (data not shown).

For combinations of idarubicin and tamibarotene, moderate synergy wasobserved for NOMO-1, Sig-M5 and MV411 (see FIG. 20). No synergy wasobserved for Kasumi-1 or OCI-M1 (data not shown).

Inconclusive results were observed for a combination of sorafenib andtamibarotene in MV411, NOMO-1, KG-1a and Sig-M5 because of the largenumber of data points outside the maxima (see FIG. 21), but no synergywas observed for that combination in OCI-M1 (data not shown). Sorafenibis a FLT3 inhibitor and we also observed synergy for combinations oftamibarotene and other FLT3 inhibitors, such as midostaurin andquizartinib, in some of the cell lines. Without being bound by theory,we believe that synergy with FLT3 inhibitors requires both high RARAand/or high IRF8 mRNA levels, as well as high FLT3 mRNA levels. This“conditional” synergy was also seen with the GCR inhibitor prednisone,which seemed to require high GCR mRNA levels as well as high RARA and/orhigh IRF8 mRNA levels. It was also observed with the JMJD3/JARID1Binhibitor GSKJ4, which seemed to require high JMJD3/JARID1B mRNA levelsas well as high RARA and/or high IRF8 mRNA levels to see synergy.

We observed no synergy in any cell lines tested for a combination of theBCL2 inhibitor ABT199 and tamibarotene. We also did not observe synergywith a combination of the EZH2 inhibitor EPZ6438 and tamibarotene.

We did, however observe synergy for a combination of the HDAC inhibitorSAHA and tamibarotene in high RARA and/or high IRF8 mRNA AML cell lines.

In addition, strong synergy was observed for a combination of decitabineand tamibarotene in HL-60 and KG-1a cells (data not shown).

We also observed synergy for a combination of the Zn fingertranscription factor inhibitor ATO and tamibarotene.

Without being bound by any particular theory, it can be hypothesizedthat non-APL AML characterized by high RARA levels, high IRF8 level or acombination of both are likely to respond synergistically tocombinations of tamibarotene and one or more of azacytidine, arsenictrioxide, midostaurin (in AML characterized by high FLT3 mRNA levels),cytarabine, daunorubicin, methotrexate, idarubicin, sorafenib (in AMLcharacterized by high FLT3 mRNA levels), decitabine, quizartinib (in AMLcharacterized by high FLT3 mRNA levels), JQ1 (a BRD4 inhibitor), ATO,prednisone (in AML characterized by high GCR mRNA levels), SAHA, andGSKJ4(in AML characterized by high JMJD3/JARID1B mRNA levels).

REFERENCES

-   1. Niederreither, K. & Dolle, P. Retinoic acid in development:    towards an integrated view. Nat. Rev. Genet. 9, 541-553 (2008).-   2. Chapuy, B. et al. Discovery and Characterization of    Super-Enhancer-Associated Dependencies in Diffuse Large B Cell    Lymphoma. Cancer Cell 24, 777-790 (2013).-   3. Tamura, T., Kurotaki, D. & Koizumi, S. Regulation of myelopoiesis    by the transcription factor IRF8. Int. J. Hematol. 101, 342-351    (2015).-   4. Yang, J. et al. Cutting Edge: IRF8 Regulates Bax Transcription In    Vivo in Primary Myeloid Cells. J. Immunol. 187, 4426-4430 (2011).-   5. Pogosova-Agadjanyan, E. L. et al. The Prognostic Significance of    IRF8 Transcripts in Adult Patients with Acute Myeloid Leukemia. PLoS    ONE 8, e70812 (2013).-   6. Sharma, A. et al. Constitutive IRF8 expression inhibits AML by    activation of repressed immune response signaling. Leukemia 29,    157-168 (2015).-   7. Smits, E. L. J. M., Anguille, S. & Berneman, Z. N. Interferon α    may be back on track to treat acute myeloid leukemia. OncoImmunology    2, e23619 (2013).-   8. Chelbi-Alix, M. K. & Pelicano, L. Retinoic acid and interferon    signaling cross talk in normal and RA-resistant APL cells. Leuk.    08876924 13, (1999).-   9. Encode Project Consortium, An integrated encyclopedia of DNA    elements in the human genome. Nature 489: 57-74 (2012).-   10. SY-1425-P003: Effects of tamibarotene (SY-1425) on proliferation    of Acute Myeloid Leukemia (AML) cell lines in comparison to    all-trans retinoic acid (ATRA)-   11. SY-1425-P006: Characterization of the RARA enhancer and RARα    mRNA in AML patient samples and cell lines

The invention claimed is:
 1. A method of treating non-acutepromyelocytic leukemia acute myelogenous leukemia (non-APL AML) ormyelodysplastic syndrome (MDS), the method comprising a step ofadministering a combination of tamibarotene and a second therapeuticagent to a subject having non-APL AML or MDS, wherein a biologicalsample obtained from the subject has been determined to have an IRF8biomarker and/or a RARA biomarker, wherein: (I) the IRF8 biomarker is orcomprises (a) an elevated IRF8 RNA transcript level relative to athreshold level that defines a dividing line between subjects whorespond to tamibarotene and subjects who do not respond to tamibaroteneand is pre-determined by analysis of IRF8 RNA transcript levels in apopulation of samples comprising a cell line representing non-APL AML, acell line representing MDS, a xenograft representing non-APL AML, axenograft representing MDS, a biological sample from a patient sufferingfrom non-APL AML, or a biological sample from a patient suffering fromMDS, wherein the number of samples in the population is sufficient toreasonably reflect the distribution of IRF8 RNA transcript levels in agroup of non-APL AML or MDS patients that is larger than the populationof samples; the analysis of IRF8 RNA transcript levels in the populationcomprises testing at least some of the samples for responsiveness totamibarotene and establishing (i) the lowest IRF8 RNA transcript levelof a sample in the population that responds to tamibarotene and (ii) thehighest IRF8 RNA transcript level of a sample in the population thatdoes not respond to tamibarotene, thereby defining the lowest IRF8 RNAtranscript responder and the highest IRF8 RNA transcript non-responder,respectively; and the threshold level is set (i) at a level equal to orup to 5% above the IRF8 RNA transcript level in the lowest IRF8 RNAtranscript responder, (ii) equal to or up to 5% above the IRF8 RNAtranscript level in the highest IRF8 RNA transcript non-responder, or(iii) to a value in between the IRF8 RNA transcript level of the lowestIRF8 RNA transcript responder and the IRF8 RNA transcript level of thehighest IRF8 RNA transcript non-responder or (b) a super enhancerassociated with an IRF8 gene; (II) the RARA biomarker is or comprises(a) an elevated RARA RNA transcript level relative to a threshold levelthat defines a dividing line between subjects who respond totamibarotene and subjects who do not respond to tamibarotene and ispre-determined by analysis of RARA RNA transcript levels in a populationof samples comprising a cell line representing non-APL AML, a cell linerepresenting MDS, a xenograft representing non-APL AML, a xenograftrepresenting MDS, a biological sample from a patient suffering fromnon-APL AML, or a biological sample from a patient suffering from MDS,wherein the number of samples in the population is sufficient toreasonably reflect the distribution of RARA RNA transcript levels in agroup of non-APL AML or MDS patients that is larger than the populationof samples; the analysis of RARA RNA transcript levels in the populationcomprises testing at least some of the samples for responsiveness totamibarotene and establishing (i) the lowest RARA RNA transcript levelof a sample in the population that responds to tamibarotene and (ii) thehighest RARA RNA transcript level of a sample in the population thatdoes not respond to tamibarotene, thereby defining the lowest RARA RNAtranscript responder and the highest RARA RNA transcript non-responder,respectively; and the threshold level is set (i) at a level equal to orup to 5% above the RARA RNA transcript level in the lowest RARA RNAtranscript responder, (ii) equal to or up to 5% above the RARA RNAtranscript level in the highest RARA RNA transcript non-responder, or(iii) to a value in between the RARA RNA transcript level of the lowestRARA RNA transcript responder and the RARA RNA transcript level of thehighest RARA RNA transcript non-responder or (b) a super enhancerassociated with a RARA gene; and the second therapeutic agent is a BCL2inhibitor, a DNA methyltransferase inhibitor, a DNA synthase inhibitor,a topoisomerase inhibitor, a FLT3 inhibitor, a folate inhibitor, a BRD4inhibitor, a Zn finger transcription factor inhibitor, a GCR inhibitor,a CDK7 inhibitor, an HDAC inhibitor, a JMJD3/JARID1B inhibitor, an EZH2inhibitor, a LSD1 inhibitor, a proteasome inhibitor, a DNA damage repairinhibitor, a PARP inhibitor, a mTOR inhibitor, a DOT1L inhibitor, atubulin inhibitor, a PLK inhibitor, or an Aurora kinase inhibitor. 2.The method of claim 1, wherein the tamibarotene and the secondtherapeutic agent are administered concurrently.
 3. The method of claim1, wherein the tamibarotene and the second therapeutic agent areadministered sequentially.
 4. The method of claim 1, wherein theelevated RARA RNA transcript level and/or the elevated IRF8 RNAtranscript level have been independently determined using fluorescenthybridization, PCR, qPCR, qRT-PCR, RNA sequencing, RNA hybridization andsignal amplification or northern blot.
 5. The method of claim 1, whereinthe subject is suffering from non-APL AML.
 6. The method of claim 1,wherein the subject is suffering from MIDS.
 7. The method of claim 1,wherein the biological sample obtained from the subject is a bone marrowaspirate or whole blood.
 8. The method of claim 7, wherein the bonemarrow aspirate or whole blood is processed to remove one or morecomponents therefrom.
 9. The method of claim 8, wherein the whole bloodis processed to yield a peripheral blood mononuclear cell (PBMC) sampleor an enriched PBMC sample.
 10. The method of claim 1, wherein thebiological sample obtained from the subject has been determined to havethe IRF8 biomarker.
 11. The method of claim 10, wherein the IRF8 RNAtranscript is transcribed from a genomic DNA sequence that encodes aninterferon consensus sequence-binding protein or splice variant thereofand specifically excludes gene fusions that comprise all or a portion ofthe IRF8 gene, and the IRF8 gene is located at chr16:85862582-85990086in genome build hg19.
 12. The method of claim 1, wherein the biologicalsample obtained from the subject has been determined to have the RARAbiomarker.
 13. The method of claim 12, wherein the RARA RNA transcriptis transcribed from a genomic DNA sequence that encodes a functionalretinoic acid receptor-α gene and specifically excludes gene fusionsthat comprise all or a portion of the RARA gene, and the RARA gene islocated at chr17:38458152-38516681.
 14. The method of claim 1, whereinthe IRF8 RNA transcript is an IRF8 mRNA.
 15. The method of claim 1,wherein the RARA RNA transcript is a RARA mRNA.
 16. The method of claim1, wherein the second therapeutic agent is a DNA synthase inhibitor. 17.The method of claim 16, wherein the DNA synthase inhibitor is ara-C. 18.The method of claim 1, wherein the second therapeutic agent is a DNAmethyltransferase inhibitor.
 19. The method of claim 16, wherein the DNAmethyltransferase inhibitor is decitabine or azacitidine.
 20. The methodof claim 1, wherein the second therapeutic agent is a CDK7 inhibitor.21. The method of claim 1, wherein the second therapeutic agent is aBCL2 inhibitor.
 22. The method of claim 21, wherein the BCL2 inhibitoris ABT199.